AU2014256400B2 - Methods, surface modified plates and compositions for cell attachment, cultivation and detachment - Google Patents

Description

METHODS, SURFACE MODIFIED PLATES AND COMPOSITIONS FOR CELL ATTACHMENT, CULTIVATION AND DETACHMENT

FIELD OF THE INVENTION

The present application is a divisional application of Australian Application No. 2009215516, which is incorporated in its entirety herein by reference.

[0001] This application claims priority to provisional application serial number 61/030,544, filed February 21st 2008.

[0002] The present invention relates to the field of mammalian cell culture, and provides methods and compositions for cell attachment to, cultivation on, and detachment from a solid substrate surface containing from at least about 0.5% N, a sum of O and N of greater than or equal to 17.2% and a contact angle of at least about 13.9 degrees, lacking a feeder cell layer and lacking an adlayer. In one embodiment of the present invention, the cells are treated with a compound capable of inhibiting Rho kinase activity. In another embodiment, the cells are treated with a compound capable of inhibiting Rho activity.

BACKGROUND

[0002a] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[0003] Cultivation of mammalian cells is one of many processes in the life and health sciences. Vessels for mammalian cell culture and analysis involving anchorage-dependent cells are often made of glass or a polymer, such as, for example, polystyrene, that frequently requires additional surface treatment to allow the cells to attach to the surface of the vessel. Such treatments may include applying an adlayer on the surface, for example, by adsorption, grafting or plasma polymerization techniques. Alternatively, the surface treatment may be via chemical modification of the vessel surface itself, which can be achieved by, for example, atmospheric corona, radio frequency vacuum plasma, DC glow discharge, and microwave plasma treatments. These surface treatments change the composition of elements and chemical groups in the surface. The particular chemistry that results depends on the surface treatment method, energy, and time, as well as the composition of the gasses used.

[0004] For example, US5449383 discloses a substrate comprising a bulk polymeric material; and a thin polymeric layer which is suitable for supporting cell growth, comprising a reorientation resistant polymer comprising plasma-polymetiasd: amide .monomer» $rdS^ih$[ amide groups for the attachmen t of cell% wherein said amidemonomers are selected from foe group of dimethyl1formamide and R!-CO~N(R2).R~' wherein E' is an aliphatic, aiieyc!ic:. or aromatic group, each of which may be optionally substituted by halogen atoms or hydroxyl groups, and E" and EJ are each independently hydrogen or an alhyl group, and wherein said thin polymer layer promotes attachment and proliferation of said ceils. f0085] in another example, EP0348909A! discloses a tr^diod.-ihrMdotheiiai^m». of a polymeric surface comprising contacting a polymeric surface with a plasma generated bom a ggipsti|::mai:SrtSl whereby said polymeric surface is modified to contain surface amino groups, and applying to said modified surface sufficient eudothcliai cells to form a confluent layer of cells on said amino gmup-containing surface without a requirement for cell proliferation. |88MJ In another example, EP00923QSA2 discloses a method for influencing the growth of cell culture in a growth media oh a mfosfoate, eharaetepaed; in that the surface chemistry of the substrate is modified by sobjeefing the surface of the substrate to a plasma, which is produced froth carbon, hydrogen, oxygen, nitrogen, sulphur, phosphorus, a: halogen, or a'compound of any one of these elements. f 0O87| In another example, CIS: 6,61.7,152B2 discloses ah. apparatus for treating a polymeric substrate surfooe eoihprising:r:(hf a gas inlet, a microwave energy source and a plasma mixing chamber, the plasma mixing chamber in Enid communication with both the gas inlet and the miemwave energy source:; (b) a dual chambered treatment area having an inner treatment chamber contained wi thin an; outer treatmen t chamber, said inner treatment chamber having an opening m fluid communication wi th said outer chamber; (c) said plasma mixing chamber in fluid commimication with said outer treatment chamber by means of m aperture; Cd) a yaeuum outlet hue attached to said outer chamber;(e) whereby said opening in satdinner treatment chamber is aligned with said aperture, saidopening being spaced from said aperture:at: predetermined distance. f 8888J In one example, US20Q3/0186903A1 discloses a polymeric substrate having a working surface upon which cells can be cultured wherein the surface oxygen content is at least 25 percent as measured by electron microscopy for chemical analysis at depth about; §0 Angstroms. )0009) In one example,, WO2G06114098: discloses a micro-sintetured biocornpatihle material for sitrgicai Implaels and cell guiding tissue culture surfaces, idle rnierostrueture of the biomaterial snr&ee is selected fopfoeune growth of undiffcrcnttated ES cells' promote neuronal differentiation of ES cells; or promote diffemntiarion of ES: cells. 10010) lb another example, Bigdeli et qL .id, EsotechnoL 133:.146-153,2008) describes a method, of adaptation and/or selection of human ES cells: to be cultivated without; differentiation under feeder-coil fee eohdldons and without prior treatment of foe solid snbstrate surface with extraceilulatmatris prole in, involving (i) changing media from:medium: conditioned by human: diploid embryonic lung fibroblasts to medium, conditioned by heouataf ehondrocytesgfii) then passaging the ceils enayoMtically from the mouse embryonic feeder cell layer to Matrigel*—treated plates, then, to Gostar5^ plates, and, finally, to PdmarialM p latest and (tii) changing back to the fo'Sl used medium again, Veri/ fow of the huntaoES cells subjected, to this method gave: rise to established cell' lilies, suggesting that this method invtovmaeiection of human ES cells to the culture conditions; flieil | Snrfece treaimen.ts that change the composition of elements and chemical groups in the surface itself have successfully been used for preparing polymer solid substrates forthe culture of many types of mammalian cells, Oowever, there are significant limitations in terms of poor attachment and/or cultivation using : certain ty pes of mammalian cells, for example, pforipotedtst^lcblt§''^dtbufoab--dfobiyo«ie..kidbby.:' CHBK) 293 cells. 10612) Graham et ak, (3, Gen. Virol 36:59-72, 1977) disclose the generation of the cell line BEK 293. 10613) HEK293 coll attachment may he enhanced by making an adlayer on the solid substrate surface, using,tor example, extracellular matrix proteins, polylysine, poiyornithine, or poiyetliyioneiminc, before adding the BEK293 cells to the culture vessel, Preparing the adlayer Is, however, dtfe-eohsnmthg, and typically results in a non-stenle solid substrate with a shorter shelf life than the bare solid substrate.

Therefore, there is a significant need for methods and materials for enhancing the attachment: of.HEK293 cells to solid substrates lacking m adlayer, f 001.4] Current reethods ef culturing pinripotmt stem cells, in particular, embryonic stem |BS} cells require complex mdture conditions, such as, for example, culturing the embryonic stem ceils on a solid substrate surface with a feeder cell layer, or on a solid substrate surface with an adlayer of ex trace! hilar matrix protoim Culture systems that employ these methods often use feeder cells or extracellular matrix proteins obtained from a .different species than: that of the sfornpells being cultivated (xenogeneic material). Media obtained by exposure to feeder cells, that is, media conditioned by cells other than imdifforeptiaied IS cells, may be used to cnlmrethe ES cells, and media may be supplemented with animal serum., $015| For example, Renbhio# it at (feature BiotechnoL· /1|;099-4ί)4«, 2000) arid Thompson. el at (Science 282 :1145-1.1.47, 1998) disclose the eplture of BS cell lines from human blastocysts using a mouse embryonie fibroblast feeder cell layer.

[001.6] fn another example, Xu at al. (Nature Biotechnology 111:971:-924,200.1) discloses foe use of Matrtgelatid laminin for treating solid substrate surfaces before foedemeell free cultivation of human BS eellsMthouf differentiation, [0017] in another example, Vallier et mi 0, Cell Sch 118:4495-4509,2005) discloses the use of fetal bovine serum for treMngaolid substrate surfaces before feeder-eeli free cultivation of human ES cells without difforentiation, |0018) la another example, WQ2f}05O14799 discloses conditioned medium for fop maintenance, proliferation and differentiation, of mammal ian cells. W02005014799 state: fofhe culture medium produced in accordance with foe present invention is conditioned by the cell secretion acti vity of mnrme cells, in particular* those differentiated and immorialked transgenic hepafocytes, named MMH (Met Murine Hepatocyte),” 10019] In anofoer example, Wanatabe et al. (feature BiotechnoL ;35 >681>0|0,200 /) state '‘a ROCK inhibitor permits survival of dissociated human embryonie: stem cells'', and demonstrate reduced dissociation-induced apoptosis, increases cloning efficiency (from, approximately 1¾ to approximately 27%) and facilitation: of suheloping after gene imtsfeiy embryonic fibroblasts as feeder cells, collagen and

Matrigelm exoacellular matrix protein, and ¥-27632 of Fashdil for inhibition of ROCK. Furthermore,dissociated human ES ceils treated with Y~2?P:2 were protected tea apoptosis in. serutm-fxee: suspension: culture. {$¢20] In another:example, Peerani di d/. CEMBO journal 26:4744-4755,2907} state “Complexity in the spatial organization of human embryonic stem eell (hESC} cultures estates iunerogeoeous mictoenvimnors^ that influence hlSC fete.

This stncly demonstrates that; the rate and 0.f .<$» bd controlled by engineering hESC niche properties. Niche: size andi composition regulate the balance between differentiaHon-indnemg and: --Inhibiting factors. Mechanistically, a. niche size-depeud.ent spatial gradient of SmadI signaling is generated as a residt ofantagohtsite:interactions between hESGs aad bust'-derived extra-embryonic endoderm 0ΝΕ), These Interactions arc mediated by the localized; secretion of bone morphogenetic protein-2 (BMP2)by ExE and its nntagonixh growth differentiation factor-3 (GDE3) by hESCs, M leropatterniag of hESCY treated with small interfering (si) ENA against GjDF3, BME2 and SmadI, as well treatments with a Rho-asSOeiated kinase-(ROCK) inhibitor demonstrate that independent control of Smadl privation can rescue the colony size-dependent diifemntiatioo of hESCs. Out results illustrate, lor the first time, a rolefbr Smadl in the integration of spatial mfermatioh and in the niche-size dependent control of hE.SC' self-renewal and differentiation/5 fttOlt) In another example, Koyanagg M etaiQ Mmroses Eos^ 2007 Sep 7 [Epub ahead Of print] ) state “Rho-GTFase has been implicated in the apoptosis of many cell types, including netimns, hut the mechanism by whichit acts is hot fully understood. Here, wd fhvPsiigete tire roles of Rho and E0CK in apoptosis during transplantation of embryonic; stem cell-derived neural precursor cells. We rind that dissociation of neural pmcnmors aetivates Rho and induces apoptosis, Treatment with theRho inhibitor €3 exoenzyme:and/or the ROCK Inhibitor Ύ-27632 decreases the amount of dissoeiation-itidueediapopiosis: (anoikis) by 20-30%, Membrane: hlebbing, w hich Is an early morphological sigh of apoptosis; cleavage Of easpafe-feand release : Of cytochrome c ftpni the mitochondria are also reduced by ROCK inhibition. These results suggest that dissociation of neural precursor cells elicits an intrinsic pathway of cell death that is at least partially mediated through the Rho/RQCR pathway. iVioreover. ife an animal iranspkniatidn model, inhibition of Rho and/or ROCK suppresses acute apoptosis of grafted cells. After transplantation, tumor necrosis factor-alpha and pro-nerve gmwtfe factor are strongly expressed around the graft, ROCK. Inhibition also suppresses apopipsis enhanced by these inRumnmipry cytokines. Taken together, these results indicate that inhibition of Rho/ROCK signaling may iOiprove survival of grted cells in cell replacement therapy.}, |0022] In another example, Yb»eda.':efi^.:p..'CbitBiol. 1.70:. 443-453, August 3, 2005) states ‘the hotmiogo^rnannnaliM^ho.'kih'^s (ROCK.3 and II) am ass umed to be functionally redundant, based largely on kinase construct overexpression. As downspeam effectors of RhoflTRases, their major substrates are myosin light chain and myosin, phosphatase. Both kinases are implicated in microfikment bundle assembly and smooth m usele copfractility. Here. analysi s of fibroblast adhesion to ibroneetin revealed that although ROOC II was more abundant, its activity was always lower than ROCK k Specificreduction of ROCK I by siRNA resulted in loss of stress fibers and: focal adhesions, despite persistent ROCK 11 and guanine triphosphate-bound RhOA. to contrast, the microfilameni cytosfceleton was enhanced by ROCK If down-regulation, Phagocytic strongly down-regulated in.ROCK If-depleted cells but not: those lacking ROCK. 1 These effects Originated in part ffoni distinct lipid-bindmg preferences of ROCK pleckstrin homology domains. ROCK II hound pbosphatidySinositol 3.4,5P;, and was: sensitive id its levels, properties not shared by ROCK I, Thereibre, endogenous R< X' K' are distin ctly regulated and in: turn: are: involved with different myosin compartments.” 1110231 Ip; another example, Harb ei ai (PbS ONF 3(8): edflOl. old 0,1371 i)oumal:.poue,O:D{?3il01, August :2008} discloses an essential; role of the: Rbo-Roek-hiypsin, signaling; axis for the regulation: of basic cell-cell communications in both mouse and human ES: cells, and would contribute to advance [sic] ip medically compatible xeao-tee environments Tor human, pluopotent stem cells, |0024j The use of xenogeneic material may be unsuitable for certain applications utilising pluripofent stem sells, Altemati^>ma,tefi.alS'.ia»y''te:u^, For example, Stojkovie or a}, (Stem Ceils;23:8SS-002,2003} discloses the use of human serum tor treating solid substrate airfiaces before feeder-cell fee enltivatfon of human ESteplIs without differentiation. f0O2SJ Ao; alternative cuitnre system employs serum-fee medium supplemented with growth footers capable of promoting foe ptoli feratkm of ES eeibe 10020] For example, Cheon eial (Bioleprod DOI:lOJ-OP^toit^prodJ05,046870; 19 Get

2005} disclose a foeder-eelFfee, serum-fee culture system in which ES cells are malnmlued in p:i»nditwo#:^P0m -replacem^Eip^di^mJuppiemented with difbfeht growth factors capable of triggering ES ceil selfuonewaL |882?) hr. another example, Levegstefo m at. (Stem fells 24:Sf?8-5?4,2006) disclose methods for foe long-term culture of human ES cells in foe absence of fibroblasts or conditioned medifofo using media supplemented with basic fibroblast growth factor (EGF). f0028j In armther example, ElSIOOSOI 48070 discloses a method of culturing human. ES cells in defined media withpur spmm and without fihrtfojasi feeder cells, the foefood comprising: culturing the stem cells in a culture medium containing albumin, amino acids,: vitamins, mraemls, at least one tmnsforrin. oirttfotsfenin substitute,: at least one insulin or insulin substitute, the culture medium, essentially fee of mammalian fetal: serum andeontaining at least about 100 ng/ml of a EOF capable of activating a EGF signaling foeepfoty wherein the grpwfh: factor:is: supplied, from a soufoeoihet than just a fibroblast feeder: layer, the medium, supported foe proliferation of stem ceils in an undiferentiated state wi thout feeder cells or conditioned medium . 180291 1« another example, 11820050233446 discloses a, defined media useful in. culturing, stem ceils, including undifferentiated primate primordial stem cells, in solution,: the media is substantially isotonic as Compared to the stem cells being cultured.. In a given culture, foe particular medium comprises a base medium and an amount of each of basic FGF, insulin, and ascorbic acid necessary to support substantially updiffotentiated growth of foe primordial stem cells. 180301 In another example, US68OO480 states “In one embodiment, a cell eulfom medium for groyring primafe-derived primordial stem cells in a subsfoutialiy undifferentiated state is provided which includes afow osmotic pressure, low endotoxin.basiumedium that is elective is» support the growth of primate-derived primordial stem eel Is. The basie medium is combined with a nutrient serum effective to support the growth of primate-derived primordial stem cells and a substrate #!eeted fo>m the group consisting of feeder cells aod an extraeeiiular matrix eompTOem derived from feeder cells. The medium further includes nonessenfiai ammo acids, an anii-oxidaoi, and a first growth factor selected from the group cousishng of nucleosidesa^ a pyravate saitfe [8831] In another; exampieeUS20050244§62 states: “In one aspect the invention provides a method of culturing primate embryonic stem cells. One cultures the stem ceils in a eultum essentially tree of mammalian fetal serum (pmferahly also essentially free of any animal serum) and in the presence of fibroblast growth factor that ts supplied from a source other than just a fibroblast feeder layer. In a preferred form, the flhiobiast feeder layer, .previously required to sustain a stem cell culture, is rendered unnecessary by the addition of snfflefent iihroblaSt growth feetor,” 18032] In another example. WO2O058h33S4 discloses a deiinedi isotonic culture medium thatis essentially feeder-free and serwmffee, comprising: a. a basal medium; b, an amoun t of basic fibroblast growth factor sufficient to srrpport growth of substantially Undifferentiated mammalian stem cells; e. an amount of insulin sufficient to support growth of substantially undifferentiated mammalian stent:cells; andd. an amount of ascorbic acid sufficient to support growth of substantially nodi fferentiated mammalian stem cells.

[8033] in another: example, WO208S86845 discloses a method for maintenance of an undifferentiated stem: cell,, said ntgihod comprising exposing a stem ceil to a member of the transforming growth faetor-beis (TGFpj&amp;mily of proteins, a member of the fibroblast: growth feetor (FGfi) family of proteins,: of memniamide fhOC ) in an. amount sufficient to maintain the, cell in an undilferentiated state fer a sufficient amount of time to achieve a desired, result. 18834] Pluripoteot stem, cells provide; a potential resource For research and drug screening.

At present, large-scale culturing of human ES cell lines is ptoblematlc and provides: substantial challenges, A: possible solution to these challenges is to passage and culture the human ES cells as single cells. Single eelisare more am enable to standard; tissue culture: techniques, such as, fer example, counting, transfection, and the like. |ί{|35] For example, 'Nicolas eta E provide a method for producing sod expanding human J3&amp; cell. Ikes from single cells feat have been isolated by Saoresceoee-activided eeli modification by Ientivirus: vectors (Stem Cells Dev. 16:109- 118, mm), |903d| In another example, US patent application US2005158852 discloses a method ‘Tor improving growth and survival of $ϊη^Ίη^^"@ηΦψθβ.ίο stem cells. The method includes the step of obtaining a single undifferentiated hES coil; mixing fee single undriferenriated ceil with an to encompass the cell; and inoculating the mixture onto feeder cells with a nutrien t medium in a growth environment”. |603?j In another example, Sidho mat (Stem Cfells Dev, 15:61-69,200^1 describe fee Erst report offeree hnhfenES cell clones, hES 31, 3.2 and 3.3, derived from fee parent line hES3 by sorting of single-cell preparations by How cytometry. tO&amp;fSj However, passage and culture of human ES cells as single ceils leads to genetic abnormalittes and the loss of pinripotency. Culture conditions are important in the mainienancc of pliiripotency and genetic stability. Generally, passage of human ES cell lines is conducted manually or with en^·malic agents such as collagenase, ilherase or dispase. ff 1139] For example, Draper et uL note the pnwmee of “karyotypic:; changes involving the: gain of chromosome 12q in three independent human e mbryoni c stem ceil lines on, five independent occasions.'’ (Mature Blotechnoi. 22:53-54,2004). ]0049J in another example, Buzzard efal state, ;iwe have only ever detected one karyotype change event,..the culture methods used may have had some bearing on our results, given dial Pbr methodsiare distinctly different fipm. those used by feest other groups. Typically we'passage human ES cells after.? days by first dissecting the colony with the edge of'a broken pipette.. .No enzymatic or chemical methods of Celtdisspciaiion am incorporated into this method. We speculate feat this may explain the relative cytogenetie resilienve of hES (human IS) cells in our hands,” (Mature Blotechnoi, 22:381-«, 2004).

[0041] In another example, Mitalipova et al. state “bulk passage methods... can perpetuate aneuploid cell populations after extended passage in culture, but may be used for shorter periods (up to at least 15 passages) without compromising the karyotypes.. .it may be possible to maintain a normal karyotype in hES cells under long-term manual propagation conditions followed by limited bulk passaging in experiments requiring greater quantities of hES cells than manual passage methods, alone, can provide”. (Nature Biotechnol. 23:19-20, 2005).

[0042] In another example, Heng et al. state “the results demonstrated that the second protocol (trypsinization with gentle pipetting) is much less detrimental to cellular viability than is the first protocol (collagenase treatment with scratching). This in turn translated to higher freeze-thaw survival rates.” (Biotechnology and Applied Biochemistry 47:33-37, 2007).

[0043] In another example, Hasegawa et al. state, “we have established hESC sublines tolerant of complete dissociation. These cells exhibit high replating efficiency and also high cloning efficiency and they maintain their ability to differentiate into the three germ layers.” (Stem Cells 24:2649-2660, 2006).

[0044] Therefore, there is a significant need for methods and compositions for the cultivation of mammalian cells, including cultivation of pluripotent stem cells in the absence of feeder cells and an adlayer, while maintaining the pluripotency of the cells.

[0044a] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

SUMMARY

[0044b] According to a first aspect, the invention provides a method to attach cells to a corona-plasma treated polystyrene surface containing at least 1.7% N, lacking a feeder cell layer and lacking an adlayer, comprising the steps of: a. Obtaining a suspension of the cells, and b. Adding the suspension of cells to the surface and allowing the cells to attach.

[0044c] According to a second aspect, the invention provides a surface that is part of a vessel or matrix when used in cell culture or analysis, lacking a feeder cell layer and lacking an adlayer, wherein the surface allows the attachment and cultivation of cells, and wherein the surface has one of the following features: contains at least 1.7% N, has a sum of O and N of at least 29.6% and has a contact angle of at least 14.3 degrees; contains at least 2.0% N, has a sum of O and N of at least 30.7% and has a contact angle of at least 18.4 degrees; contains at least 2.1% N, has a sum of O and N of at least 30.2% and has a contact angle of at least 17.4 degrees; or contains at least 1.8% N, has a sum of O and N of at least 28.2% and has a contact angle of at least 18.8 degrees.

[0045] In one embodiment, the present invention provides methods and compositions for the attachment, cultivation and detachment of cells to a solid substrate surface containing from at least about 0.5% N, a sum of O and N of greater than or equal to 17.2% and a contact angle of at least about 13.9 degrees, lacking a feeder cell layer and lacking an adlayer.

[0046] In one embodiment, the present invention provides a method to enhance the attachment of cells to a surface containing from at least about 0.5% N, a sum of O and N of greater than or equal to 17.2% and a contact angle of at least about 13.9 degrees, lacking a feeder cell layer and lacking an adlayer, comprising the steps of: a, : Obtaining a suspension:ofcells, bv Treating the suspension of cells with,It least otT#:.ii?Ojst^©utitii Mccted &amp;bifi,the group consisting of: a compoimd icapable of inhi biting: Rho kinase activity, andu: compound capable of iobibidog llho: activity,, and! e. Adding the suspension of cells to the surface and allowing the cells to attach. |'O047] jit one embodiment. the cells are maintained in cul tore after the cells:attach to tile surface, In an alternate cvobOdtmOnt'fhe at least one compound, ktemoved. (0048] Inone embodiment, the ceils are detached from the surface by retnoying the at least opO: compound.

[0049] in .one embodiment the suspension of cells is a suspension of clusters of cell&amp; In an., alterpate emhodixneut, die suspension of cells is: a suspension of single cellA 1111151)] iaoae emboidiment, the cells are plnripotentstem: eelis..: I» an. alternate embodiment the1 cells:are stem cells. 1011511 la one embodiment, the. present invention provides a method to enhance the attachment of cells to a surface containing from at least about 0.9% N, a sum of O and M of greater than or equal to 22.3% #d a contact angle ofat least abbot 13.9 degrees, lacking a feeder cell layer and lacking an adlayer, compristug the steps of: a. Obtaining a suspension of cells, and h. Adding the suspension of cells to the surface and allowing the cells to attach.

BRIEF DESCKI FT! ON OF THE BRAWLS

[0052] Figure 1 shows phase coutmat mierographs {4¾} of cells of the human E$: cell line II1 that weiopassaged twice as clusters with. LIBER ASE on surface modified plates 2,, 3 or4. Images of cells of the: human E;S cell line HI, cultured on plates treated oath a 1:30 .dilution ofMatrigell^Nuneion Delta ^ plates are also shown.. |0#53J Figure 2 shows the effect::of 111 pM Y~2?632 on the aitschmeut ofburnan IBS cells to surface modified plates. The figure shows phase contrast micrographs f4s) of cells of the human ES cell line H I that were passaged twice as dusters on surface modified plates 3 and 4. Cells were then passaged onto surface modified plates 2,3 or 4fon MEF conditioned rnediOfoeeniaiMng 10 pM ¥-27632. Cells were cultured for four days prior to taking the photographs. Cells etthured in the absence of ¥-27633 were included as controls, f00$4} Figure 3 shows a schematic of th e of compotmds ou human IS ceils cultured on the surface modiSM plates of the present irweution. Celts of the human EiS cell Hue 131: wesifoqms&amp;go&amp; four times·as clusters with L1BERASE treatment on surface modified plates % or 4, and cultured in MEF conditioned ntediusn. Cells were treated for the first two days after passage with either IQ uM of the ilho Kinase iuhifiitop ¥-27632, or with 0.5 ng/mi of the Rho inhibitor, a cell permeable form of estoenuyme C3 transferase. Cells font were treated with the Rho lOuase iuhihiior, ¥-27632 and were thereafter treated for the first two days after each, passage with ¥<-27632 oa surface modified plate 3 are referred to as ‘Wh Cells that were treated with, the Rite Kinase inhibitor, ¥-27632 and were thereafter treated for the first two days after each passage with ¥-27632 on surface modified plate 4 are referred to as ‘C#'. Cells that were treated with the Rho Inhibitor for two days and were then treated with: the Rho Kinase inhibitory ¥-27632 for; two days after each passage and thereafter treated for the first: two days after passage with. ¥ -27632 on surface modified:plate 3 are referred to ^ 'TssC, Igtslls that were treated with, the Rho inhibitor for two days and were then treated with ihe Rho kinase ihlithiior, ¥-27632 for two days after each passage and thereafter treated for the first two days after passage with ¥-2763:2 on surface nmdlfiod plafo. 4; arc referred to as“ls":. |0O5$j Figure 4 shows the expression of markers associated with piuripotency and humatP.iB§'C48rhfoMed dofoMmg to the ppMOepI outlined in Figure 6 as determined by qET~P€R.

10056) Figure 5 shows the expression of pluripofoucy Markers in cells of th e human ES cel I line HI as determined by flow cytometry at passage 4 fod), passage 9 (p9), and again at passage ffo 1.1, or 12 (pill, pi 1, or pi 2). |0ft57J Figure 6 shows immooo-fiuoreseent images of cells of the human ES cell line H I. were passaged serially as clusters with LISEKASE treatment on surface modified plate 4, ami cultured iaMEF conditioned medium. Expression of proteins associated with markers of piunpotcncy was detected in cells cidturcd for I1 passages on surface modified plate 4. Cells were treated with 10 μΜ Y-27032 for two days after each passage. f 0058J Figitre 7 shows the ability for human ES cells to form definitive endodefrn after euliure on surface modified plates:, Cells of the human ES cell line H! wore passaged I t times as cinders with. LIBERASE treatment on surface modified plates 3. or 4 and cultured, in ME!7 conditioned medium. At passage: SfpS): and again: at passage 1:0 or II (pi 0:-1.1) Cells were irea(ed %dth: DMEM:F12 media containing 0.5% BBS, 1.00· ng/ml Actiyin A, and 20ng/m) Wh|3a for two days and foctf treated with B:MEM;:FI2 media containing 2% PB:S and 1.00 ng/ml, Aetivin A for three: mote days. The y-axls on the graph shows foe: percent positive C2CCR4 cells obtained, 'by-flow cytometry.

See also Table 5:. fOllSO] Figure 8 shows the ability for human ES cells to form pancreatic endoderm after culture on surface fopdlBed plates.. Cells of the human ES cell line HI were ptrfsaged eight times as clusters with ilfillBRASlvircatinent: on. sutfeeemodifled plates 3, or 4 and cultured in MEF eohdlfidned. medium. At passage :8 (pS) ceils were sub|eeted to diffomntsation to definitive endoderm bv treatment with DMEM:P12 media containing 0.5% BBS. 100 ng/ml Aetivin A, and 20 nglml Wnt3a for two days and Own treated wifhBM:EM:FI2 tptdia eontatmhg2% FBS and 100 ng/ml AetMn A for three more days, lire ceils wem foen further diffifcntlafed fo embryonic foregot with four days of treatment with DMEMtE 12 media eoniaining 2% FBS, 100 ng/ml FGF-10, and 1 pM oyciopamihe-KAAD. The cells were then differentiated to pancreatic endoderm with four days of treatment witfi'©MEM:F12,m<^i».ooBtammg 1% B-27* 100 ng/ml FG.F-10, 1 uM cyelopaniine-KAAl) and 2 μΜ retinoic acid. Cells were stained by iunnunoflnomseenee for PDX-1 (green) and B-cadherin (red) and total cell number was identified by Hoechst dye (blue). f 1MM0] Figure 9 shows the ability of human ES ceils cultured on. surface modified plates to form embryo id bodies. fOOdtJ Figure 10 shows the fcaryoiypeof human ES cells cultured on surface modified plate 4. IIMI62} Figure I 1 shows the effect of treatment with Rho kinase inhibitors (Y-27632 front EMI) bldseience% Y-27632 fern Sigma, Fasudil, and Hydroxy&amp;sudil) on the attachment of human BB cells to surface, modified plates. Cells were cultured in medium ..containing: the,indicated. compounds, at the concentrations: listed, for three days. Cells were stained with crystal violet and. Images: taken. |6§631 Fi gure 12 shows the dose-response of Y-27632 on the attachment of human FS cel is to surface modified plates. Yarioua eoneeuttatidhs of the Rho kinase inhibitor, Y-27632,: was added to the cultures at a, specified concentration. f|ivl ,.2,4, or 10 μ.Μ Y-27632) -for the first day. The cells were then maintained from day 2 onward iit media containing 10 μΜ Y-27632 with daily media changes; for five days, Media.: wax rernowjdtfom the plates on day five and the cells: were stained with: 0-,334 crystal violet, and images taken:. ||I64] Figure-13 shows the formation :of human .'ES cell colonies four da> > after passage onto surface modified .plates 2-,% or d with or without .10 of μΜ Y-2/632. 1111651 Figure .14 showa the formation of human ES ccil coIonics four; days after passage onto Matfigel^ heated plates with or without I θ μ.Μ Y-27632. fS0661 Figure IS shows the difference between: eon tinnal and intemnttent treatment of human ES cells with Y-27632, on attachment of cells to surface modified plates. 109671 Figure 16 depicts images of cells from the human ES cel! Sine 119, that were passaged as single cells. seeded on fo surface modified plate 3 in MEP conditioned media containing (B) or with out (A) 10 μΜ Y-27032. The images were taken 24 hours after seeding. f OOSRj Figure 17 depicts the expression of markers associated with pluripoteney in eel Is from the human BS oell line B9, that were passaged as single: cells for 5 passages, using TrypLETM Express, and plated, onto surface modified plates 3 and 4, with or with, out 10 μΜ ofY-27632 (Y). The pluripoteney markers are listed:: on the x-axis and the percentage of positive: cells is shown, on the y~exis, 161691 Figure 18 depicts the total cell number of cells fibm the human ES ceil line :119, that were passaged as single cells, plated onto surface raodified plates 3 and 4. The effect of 10 μ.Μ of Y-27632 (Y) on ceil number was exarnined on. ceils passaged on Matrigeir^ and-bells passaged 10 times on the surface modified plates («climated, A), The different cell eopditkms are listed on the x~axix arid the tmmher of ceils d ivided Ip 10% shown in the y-axis.

[80701 Figure 18 depicts the rate of growth of cells ten the hiuftan ES eel I fine H9, that were passaged as single ceils on MatrigeFM treated plates prior to the study. Cells were seeded at 10%πΥ and cultured iu ME F conditioned media with or with out 1.0 μΜ of Y-27632 on surface modified plates 3 and 4. The y-axis shows the number of cells collected 2, 3 or 4 days after seeding (divided by 184).

[0871| Figure 20 depicts the rate of gpwth pf cells from the human ES ceil line H9, that were passaged as single cel is tor 18 passages on surface modified piates prior to the study. Cells were seeded at It^fcrn" and Cultured in MEF conditioned media with or with out 10 fiM of Y-27832 on snriaee modified piates 3 and 4. The y-axis shows the number of cells collected 2,3 Or 4 days ate seeding (divided by 10% [80721 Figure 21 depicts linages of cells fiom thehurnan ES ceil line 08, that were passaged as single ceils, seeded on to surfhee modified plates 2»4 and 13 in a 9E~weli format. The MEF conditioned media contained 10 μΜ ofY-27632. images were taheh 48 hours ate seeding, 180731 Figure 22 shows the ability of cells from the human ES ceil line M9, that were passaged as single cells, seeded on to surface modified plates 3 and 4 to differentiate; into definiti ve endoderm.. Tb e ex tout of formation of definitive en doderm was determined by measuring;€2£CR expression by flow cytometry, The effect of 1§μΜ; ¥-27832 on the formation of definiti ve en doderm was investigated. Cells were treated with Y-27632 during expansion. Ceils expanded and diflirentiated on MatrigeliM were included as a control. The y-axis shows percent positive CXCR4 cells obtained by flow cytometry.

[08741 Figure 23 shows the ability of ceils from the human ES eel! line B9, that were passaged as single cells, seeded on to surface modifiedplMes 3 and 4 to differentiate into pancreatic eudoderrm Cells were plated onto the suf&amp;ee modified platey arid cultured In MEF conditioned mediumcontaiuing 10 μΜ Y-27632, and passaged 8 fees on, the surface modified plates prior to d|ff%re«tiat£oi>> The y~a.ms slmws the fold increase of pancreatic difiemmiadon marker expression (Ngn3, Pdxl,4nsnlin):fiy q-PCR at the posterior fhregut stage (PF) and the hormone expressing endocrine ceil stage (EN), 19|?I| Figure 24 shows the attachment of human ES ceils to surface modified plates.

Passage 50 H9 human ES ceils were plated at a i;2 dilution on Sur&amp;ees 3 and 4, CellBtMP®, and PnraatiaiM. Media was removed fen the plates 24 hours after wirp stained with. 0,5% erysiai violet, and images taken. Arrows indicate colonies, jOOTdj Figure 25 showsdhe attaciiment of human IS ceils to surface modified: plates.

Passage 50 H9 human ES cells were plated at a 1:2 dilation on Sur&amp;eesS and 4, GellBFRD^V and Primaris’*’4 in thepresence of varions eoncetarations 0;f ¥-27632 (0, 1,2, 4,, 10 and 20 mieromolar). Media was removed from the plates 24 hours after plating and the ceils were stained with0,5% crystal violet, and images taken.

Colonies are dark spots on the well. Arrows are tintreated wells, [00771 Figure 26 shows the attachment of human ES ceils to surface modified plates.

Passage 53 H9 human. ES cells.: were plated^ at a 1:3 dilution on Surfaces 2-4 and 13, Ceil;B!^D'!^Van^ :ftr*:^:a^81^ &amp; the absence or presence of Y-27632 (0 or 20 mletoinplar). Media was removed, from the plates 4g hours after plating and the colls were stained with 0.5% crystal violet, and:images taken. Colonies are dark spots on the well Arrows are used to highlight colonies on. the untreated wells. |M7Sj Figure 27 shows the first attempt (October) and second attempt {December) to attach human H9 ES cells to spriaee modified plates 14 and 15 and an attempt to attach human HI ES cells to sur&amp;ce modified plates 14 and 15, Passage 42 and passage 53 FI9 human ES, and passage 57 HI human ES cells were plated at a 1:2 or 1:3 dilution to the modified surfaces in the presence of 20 nheromolar Y-27632, Media was removed: fern the plate 24-411 hours after plating and the cells were stained with 0.5% crystal violefiandImages taken. Colonies aty: dark spots pn the well. Arrows are used to highlight colonies on the plates. i ($79) Figure 28 feaman BS cells to asurface modified plate 4 in defined media*·.rnTeSR1 u. Passage SCI -H9 haman E§:cells were plated at. a 1:2 diltotoa to the nfodifiedMirfeees in the absence or presence o£¥-2?632:(9 at 20 miemmniar|in wclisA^-were-iiiiJireated 'or$$8ted with piMsiBsfb.1% gelatin, 2% BSArf).34fog/£nf tot ifoSlsgpt 1, 1:1000 dilbfod Matrigeh^, or 1:5000 diluted Manage!iM). Media was removed from the plates 48 hours after plating and the cells were stained with 0:5% crystal violet, and images taken,: Colonies are dark spots on the well, fiiStt j Figure 29 shows the water contact angles of surface modified plates measured:over 11 weeks using the static sessile drop method. The first measurement was dose one week after surface treatment and sterilization. Each data point represents the mean contact angle (one measorement on each of 7 drops). The eontaet angles on Nuclon Delta af and CeilBlND^platos were experimental conditions as Surfaces 1-4 and 13, but the surface treatrneat and sterilization was done more than 12 weeks bofi^''iiddlt#'Mea§dtotMbttBsctetDe1ite?M'^':w^steri!ipgd one week before the first measurement).

[.0081 | Figure 30 shows the density· of negative charges on surface modified plates measured as reactivity of surfaces wi th positively charged crystal violet. Three samples of each surface were iesied,: and absorbance measurements on. desorbed crystal violet from, each sample were performed in triplicate, Mean and standard deviation of nine measurements are given, [00821 Figure 31 shows the effect of solid substraie surfaces and Y-27632 on/atiaehfeem and gtowth:0f HEK293 cel Is: in chenieally defined, serum-free Pro293a-C0M™ medium. (A.) or:'EMfiM medium supplemented with ,103-¾ fetal bovine serum. (B), HBK293 cells were, seeded ip 9fovcelfplates with CeUB 11413m sorfe.ee, Nuadon Delia:*u surface or Surface 4, Theoumber of HE$*293 cells attached to these surfaces is fcwB as a function of culture Conditions and eobcentraiion of Y-27632,; Ceils received either: (i) 96 hours of constant treatment in culture: with Y-2763.2 i;Y~2:7632 96h on); or fit) 48 hours ofeonstant treatment in culture with Y-27632 followed by a change of rnediuni and: then 48 hours in culture·without Y-27632 (Y-27632 48h on/48h. off), MEK293 ceils cultured without Y-27632 in the medium. (Mo Y-27632) Were handled the santo way as cells enitured with Y-27632, that is, for either 96-hours without a change of medium, or with .a...change of medium after 48 hours. ¥-27632 crdtanced atmchomutof HEK293 cellson Surface 4 andihe €elfBiMDs>t surface wheu applied at concentrations of 2.0 and 5.0 μΜ. Removing Y-27632 after 48 hours of incubation msolied in detachment of a significant number of cells from Surface 4 and the CellBllSB1** surface. Mean and standard deviation, of three rueasurements are shown. 1:0883} figure 32 shows the effect of solid substrate suifg^^odi3Rii©''kinasie inhibitors Y- 27632 and 11-1152 on growth of HEIC293 cells in EMEM medium supplemented with 10” ·. fetal bovine serum. HEK293 cells were seeded in Muhidlsh 24-well plates with either Surfaced {&amp;) or a adtMreated (but gamma ift-adidted; 25 kGy) poiysfyfene surface (B|. |8084| Figure 33 shows the effect of fi~ 1152 and Surface 4 on ME093-eeil attachment and morphology. 11112293; cells were seeded in ynItidish l2-weSI plates in EMEM medium supplemented whh 10% fetal bovine serum and If-! 152. and incubated for 67 hours in anuiuidthatedj in-incubator microscope: Growth curves in A and photomicrographs In 31 show the generaf eftect of H-l 152 on BEK293-ee!i attachment and growth .on femlsce '4, ;and the effect of a change of medium on BEK293-cel! attachment and morphology on surface 4 in the presence or absence of B-1I52, fOOSSJ Figure 34 shows growth: curves over 3 passages fer HFIS293 ceils grown on surface 4 and Nuneloo Delta ^ suriaee iu the absence or presence of 2 J μΜ Y-27632. HER29&amp; cells in EMEM medium, supplemented with .1..0% fetal bovine serum were passaged 3 times by trypsinkallon. |8086j Figure 35 shows the effect of Rho kinase inhibition oft the attachment of cells of the human embryonic mere ceil line HI to surface modified plates 4,18 and .19, and Frimaria! M. Wells Ά&amp;Β were eontixh wells on ail surfaces. Wells C&amp;D contained 1 ΟμΜ Y-27632. Wells MW contained 3pM Hfl52-glycyi WelkG&amp;G contained ΙΟμΜ fill 52~glycyf. 108871 Figure 36 shows the effect of ifho kinase inhibition on the attachment of cells of the human embryonic stem cell line ill ioMrlaee modified plate 30. {—}no treatment, (RI) == 3 μΜ HU52-glycyL (MO)™:: adlayer of 1:30 dilution of MATRIGEL, (MG+Rf) - adlayer of 1:30 dilution of MATRlGBL -i· 3 μΜ HI l§2-giyeyi, (0PS| Figure 3? shows the effect of Rho kinase inhibition oft. the attachment of cells of the human eniBr)®nie: sterfi::eelI.; line HI to surface modified plate 31. (~~) - no treatment. (R]) « 3 μΜ H1152-glyeyi. (MG) :::: adlayer of M^ATRIpEl:,· (MGTRI)- adlayer of 1:30 dilution of MATR.1GEL + 2 μΜ Hi 1 Sl^gtyeyl:, |!ΙΜ9) Elgin® 38 shows the effect of Rbo kinase inhibition on. the attachment of cells of the hitman, embryonic stefti cell line Η1 to surface Modified: plate 32. (-) - ho treatment, (Rf). - 3 μΜ Η1152-giycy I. (MG):: adlayer of 1:30 dilution of MATRjGEL, (MG+Rl) - ad layer of ) :30 dilution of MAIR1GEL - 3 μΜ HI I52.giyeyl

Figure 39 shows the effect of Rho kinase inhibition oh the: attachment: of cells of the human embryonic stem cell line ΗI to snrfece modified plate 33'« (—) ~ no. treatment (RI) - 3 μΜ' HI 152-gfycyt (MG) - adlayer of 1:30 dilation of MATRJGEL. (MG+fllji ::ii adlayeref 1:30 dilution of MATRIGEI, r 3 μΜ HI 152~glycyl. 10991] Figure 40 shows the effect of life) Mease inhibition on the attachtnent of cells of the human embryonic stem cell line H i to surface Modified plate 34,: (—) - no treatment, (RI) - 3 μΜ B11 S2~glycyf (MG) - adlayerof 1:30 dilution of MATRIGEL, CMG-t-Ri):::: adlayer of 1:30 dilution;of MATRIGBG 3- 3 μΜ: HI l:52-glycy|, 100:92] Figure 41 shows the water contact:angles of suritce modified plates measured: over 40 weeks using the static sessile drop method, f 911931 Figure 42 shows the water contact angles of surface modified plates usmgthc static sessile drop method, (0094) Figure: 43 shows the density of negative: charges on surface modified: plates, measured as reactivity of siirfeecs with positively Charged cry^at violet f 0095] Figure 44 shows the density of negati ve charges on surface modified plates 4, 22-24 and 29 measured as reactivity of surfaces with positively charged crystal violet.

Three samples of each surface wem tested, and absorhatmc rneasttremcirts on desorbed crystal violet from, eaelisample were perfermed in triplicate. The negati ve charge density for surfaces 4,22-24 and 2$ was normalized to foe -negative: charge deasltyof the Nutffifon Delta!M sorfto. Mem aasl standard deviation offline mcaxuremenIs are given.

DETAILED DESCRIPTION 180961 For clarity of disclosure, and mot fcy way oh limitation, the detailed description,pf the invention:is: divided into the following sifoseeitons that describe or illustrate certain. foatamSjfonibodiments or applications of the prpfonfinveotiom 100971 "Adlayer” as used herein refers to a layer that is formed on a Airfoeefof a solid. substrate, by attaching molecules to the: surface by either covalent (also known as grafting) or non~eovaIent (also known as adsorption) bonds. Molecules used M making .ah adlayer can, for example, -NiiMcihi may include, for example, extmcelkdar matrix pmteins, amino acids and the like, and non·· biological molecules, sucllas, forexafople, polyetbyleneimiue, 10098) "fkeell lineage” refers to cells with positive gene expression for the transcription factor FDX-1 and at least one of the following transcription footers: NGN-3, Nkx2,fo Nkx6.1, bieuroD, ls|“l, HNF-3 beta, MATA, Pax4, and Paxi. Cells expressing markers characteristic of the p cell lineage include β cells, [80991 “Cells expressing inarkefofohamcieidsile of the defnidve endOdenn lineage” as used herein refers to cells expressingnt; least, ore of the following markers: SOX··;?. GAIA-4, HiNF-3 befo,: GS:C,:Cerl, Nodal, FGF4, Braehyury, Mix-like homeobox protein, FGF-4 CD4&amp;, eomesodermin (EDMES), DKK4,PGF47,, GAIA-6, CXCR4, C-ISit, CD99, or QTX2:, Cells ex pressing: markers characterlsifo of the definidve endoderni lineage include primitive stexfo precursor cells, primitive streak; cells, mesendddefm. ceils and definitive endoderm cells. 181001 ‘Dells expressing markers characteristic o f the pancreatic endode rm linea ge” a s used herein reforsfo cells expressing at least one of the following markers: FDX-I, FINP-Ibete, PTF-l alpha,/FfNF-fo or HB9, Cellstexptesshtg the pancreatic cpdodeimlineage include; pancmatic endoderm cells. 10181] “Ceils expressing markerscharacteristic of the pancreatic endocrine lineage” as used herein refers^-ceils: exptxxssihg at ieast one of the ftllowmg markers; NGN-o, NetrmD, Islei-I, PDX~ L ΝΚΧδ,ί, Pax-4, Mgn-3, of FTF-l alpha, Ceils expressing markers ,cN^^emdc0^:^|^dre^.;en.4bciiMlm^g$:%cIeido pancreatic endocrine cells, panen^tic hormohs expressmgceils.and pancreatic hormone secreting cells, and cells of the β-cel! lineage. 10182] “Defeitlve endoderm” as used herein refers to ceils which bear the chufectcdstics of cells arising from the cpiMast during gastraiation and which form the gastmintestinal tract and its derivatives. Definitive eododerm ceils express the following markers; CXCIM, BMF-3 beta, DA]fA-4, SOX- l'2*C<a6®ru^. DTXI^g^secoi^. c-Kit, CD99. and MlxD, fill 83] “Extracellular matrix proteins” refers to proteinaceous moieenies normally found between cells in the body or in the placenta. Extracellular matrix proteins can be deri ved from tissue, body Suidk,such as, for example, blood, or media condifioned by non · recombinant cells or recombinant, ceils or bacteria, f 0184] * "ExtraembryOnic endodemf ’ as used herein refers to a population of cell s expressing at least ope of the following markers' SOX··?, AFP, and SPARC. |0105] 4 ΉΕΙ£2:93 cel ls” refers to a cell line generated by transformation of a culture of bormai human, embryonic kidney cells as described by Graham et ui. 0, Gen, Virol. 36,5¾¾ 1.977), and any cells derived from this parent cell line. fill0b] "Markers’'· as used; herein, &amp;rp npelelc aeld: or polypeptide molecules that are differentially expressed in a eell pf interest. In this context, differential expression, means an increased level for: a positive marker and a decreased: level for a negative marker. The detedtahlC: le vel of the marker boclele acid or polypeptide is sufficiently higher or louver in fee cells of interest contpared: to other cells, such that the cell of interest ean be Mattihed mfe. distinguished ffetn other cells using: any of a variety: of methods known in the art- 10107] “Matrix” as used herein twfers to aS-dimensional support: :to which cel ls May aback (0108J “Mesendoderm cell” as used teem refers to a cell expmssing ai feast one of tbs following markers: €B4|5 edffiesodefmift (HOMES), SOX-1~, DKK4, HNF-3 tea, gsc, fgfo7, GATA~6: |0100] “Pancreatic endocrine cef!” or “pancreatic hormone expressing cell” as used herein, mfte to a cell capahje of exprekrihl «t least one of thrifollowing hormones: insulins glucagon, soiaatostatin s and pancreatic polypeptide, (0110| “Pancreatic hormone secreting cell” as irsod herein refers to a ceil capable of secrefing; at least one of the following hormones: insulin, glucagon, somatostatin, and pmtereatic polypeptide, |0111 j “Pre-primitive 8fc^.k£eiP? as osedheteim refers: to a cell, expressing at least one of the following markers: ISfodal, or FGF-B, [0112| “Primitive streak cell** as used herein refers to a cell expressing at least: one: Of the following markers; Braehyuty,(Mk4ike hotaeohox protein,, or FGF-4, (0113) “Surface” as used herein refers to the outermost layer of molecules of a solid substrate vessel or matrix intended for use in cell euliuio or analysis, The elemental composition, the roughness, and the wktability of the surface can he analped hy X-Ray Photodeetron Spectroscopy (XPS), Atomic Force Microscopy (AFM), and contact angle measurement, respective!y, [0114) “Surface modified plate refers to a vessel containing any oneoEsurfaees 034, described in Examples 16» 17 and 26, or plates con taining Surfeees that are: sold under the tmde narues Nunefon OehaSM, GostariM, FalconSM, CelMlMD^ and PrimarislM. The vessel can, tor example, be made of a poly mer, such as polystyrene (PS), cyclic olefin eopplynier (COG), polycarbonate (PC), poiymethyl methacrylate (PMMA), or styrene acrylonitrile copolymer (SAN), 101 IS) Stem ceils are undifferentiated eel Is defined by their ability at the single cell level to both, selfirenew and, diilemtmate to produce progeny cells,. Including self-renewing progenitors, non-renewing:progenitors, and terminally dlfiemntiated cells, Stem cells ate teo oitameferiied by their ability to differentiateM yfofe into functional cells of various cell lineages bom multiple germ layers (endoderm, mesoderm and ectoderm), as well as ia:giys rise to tissues oftpdiple germ, layers following tm^tataiion and to contribute substantially to most, if not ail tissues felfowirig injection into blastocysts, f$!t4j Stem, ceils are: classified fey their doveiopmental poteotlai as; (i)totipotent, meaning able to give rise to all embryonic and ex'traei»fe^nieM!types;;^fi^!iMpotSM, meaning abM to give rise to ail embryonic ceil types; Ciii):-riiiiuMpoteritt^.iea^^xtitig. able to give rise to^subset of ceil lineages, but all within a particular tissue*organ,: or physiological system (for example, hematopoietic stem colls (HSC) cafe produce progeny that include BSC (seif- renewal), blood cell testticted oligopoienl progenitors and all cell types and elements (e.g.,: platelets) that am normal components: of the blood); (iv) ofigopotent, meaning able to give; rise to; afoore restricted subset of eel! lineages than mnltipoteot stem cells; and (v) unipoteni, meaning able to give Bsc to a single cell Uncage (e,g,, speonatogetuc Stem ceils); |0117] DiffercntiatiOfe isfefee process fey which, an unspecialized fbaeommitted’) or less specialized: cell acquiresthe features: of a specialized cell such as, for example, a nerve Ceil or a muscle cell. .A^l^rentiatfeii'or difforefefeatiou-induOcd cell ;is ofee tirat has taken on a mom specialized (’foomnutted") position. wifein the lineage ofa cell Itoe term, committed”, whoa, applied to the process of diffofeniiation, refers to a cell that has proceeded ia toe differentiation pathway to a point where, under normal cfrcumstaoces, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, fejider normal into a diffettott cell type or revert to a less differentiated eel! type. Dedilferentiation retom to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell As used hereto, toe tour is, which cells it came bom and of a ceil places the ceil within a herediiafy sdierne of development and different! attorn A line^e-specific marker rotors to a charactoristic specifically associated with the phenotype of ceils ofa lineage of interest and can be used to assess the differentiation of an uncommitted, cell to the lineage of interest, fill 18] Various terms are used to describe cells in culture, "Maintenance” refers generally to cells placed ip a gmwto mediufer under conditions that facilitate cell growth and/or divisjon: that may or may not result in a larger population. of the cells. "'Passaging'’ refers to the: pmcess of :remm%g the ceils from one culture vessel and planing them m a second i&amp;hdiitons that facilitate cel! growth and/or division.

[01191 A specific: populationpfteells, or a cell line, is sometimes referredi to or characterised by the number of times it has been passaged. For example, a cul tured cell population that has been passaged ten. times may be referred to as a;f 10 culture, ΊΜ primary enlt:0mj.sthat::is> the first culture toUowmgthe isolation of cells ftem tissue, Istoesignatod PC. Imiiovtdng the first subculture,: the cells are described as a isecoadary culture (P1 or passage 1}. .After, the secoiid stfttettllure, the ceils become a tertiary culture (F2 or passage 3), andiiso on, It willhe undetatood by those: of skill in, the art that there max? be ntuny .population; doublihgs during the period of passaging;: thefeftsre the number of population doublingsof a eultane is gmater than the passage .number. The expansion of cells (that is, the number of population doublings) during the period between; passaging depends on many factors, including but not limited to the seeding density* substrate, medium, growth condition^ and: time between passaging, [01,29] Ip, one embodiment, the preseat invention provides a method to enhance the aihtchmehl of cells to a surface containing1 from at least about ¢1,9¾¾ |Sl, a snm of 0 and N of greater than or equal to 22,35¾ and a contact angle of at least about 13,9 degrees, lacking a: feeder cell layer and lacking an adky@rs.oomptfeing.'dte:ste|^ ©ft. ai Obtaining a suspension of bells, and: b . Adding the suspension of ceils to the surface and allowing the cells to attack, 10121 ] in one embodiment, tire pfeseat invention provides a method to enhance the attachment of ceils toa surface containing Iforn at toast about 0.5% M, a sum of f) and N of greater than or equal to 17.2% and a contact angle of at least about 13,9 degrees, lacking a feeder cell layer and lacking an adlayer, comprising the steps toft a. Obtaining a suspension of ceils, b, Treatfeg the suspension of cells with at least one compound se tocfedilom the group consisting oft aeompound capable of inhibiting Rho kinase activity, and a compound capable of inhibiting Rho activity, and e. Adding the suspension of ceils to the surface and allowing the cells to attack. |0122J liiiOiie embodiment tite suspension of celts is .a suspension of clusters of cells, 'in an alternate embodiment, the suspension of cells is a suspension of single cells, 10123} in one embodiment,. hie cells are plaripoient stem cells. In an alternate embodiment, the cells are stern cells. f 0124} in one embpditaenf the surtece has an adlsyer. In one enibodlmenf the adlayer is an extracellular matrix component, such as, Ibr example; those derived from basement metnferane or hlat may term, part of adhesion: molecule receptor-ligand couplings,. 'In one emhodiment, the adlayer is made from MATRIGBL (Beeton Dlekensonf, MAlllIGEL is a soluble preparation from Engelbreth-Holrn Swarm tumor ceils that gels at room temperature id item a reconstituted basement membrane. The proteinaceous adkyer may also be: formed from, laminin, fibroneeikp proteoglycan, entactiigheparan sulfate, and the like, alone or ill various eomhinations. f0i2S| In oneiembadintent, theeells am rnaia:iaihedin .culture after the cells attach to the

Sprflee. In ah alternate embodiment the at least one compound is removed: after the cells attach to the surface,, hr one etnbodimenh tire cells are detached from the surface by removing the at least one compound, 101.20} In one embodiment, ..the suspension of ceils is treated with at least one compound capable of inhibiting Rho kinase activity. In an alternate embodiment, the suspension, of ceils is treated with at least one compound capable of inhibiting Rho activity. In an alternate embodiment, the suspension of cells is treated with at least one com pound capable ofinhibiting Rho kinase activily and at least one compound capable of inhibiting Rho activity, 10127} The at least one compound capable of inhibiting Rho kinase activity is selected from the group consisting ofr Y-27632, FasudikaMFiydrQxyfasudib |0128] in one embodiment, the at least activity is Y-27632. 10120] The at least one compound capable of i nhibiting Rho kinascTaetivity may be used at a concentration, from about (kipM tp about 100μΜ> In one embodiment the at least one compound capable of inhibiting Rho kinase activity Is usedat a concentration of about ΙΟμΜ.

[0130} is one embodiment, the at least one compound capable ofInldbstmg Rbo activity is a Rho GTPase inhibi tor.

[0111 | fn one embodiment, the at least one compound capable of inhibiting Rho activity is exoenzyme €3 Transferase, [9132] The at least one eomponnd capable of inhibiting Rho activity may be used at a

concentration from aboui'0,9l|.tg/m.l to about Spg/rnt In one embodiment, the at least one cbmponnd capable of inhibiting Rho activity is used at a concentretbh of about O.fpgfinL

Surface Modified Plates 10133] Surfeee modified: plates suitable tor use in the present invention may be vessels whose surfaces have bead modified to contain fiom at least about 0,5% 1¾ a sum of O and N of greater than or equal to 17.2% and a contact angle of at least about 13.9 degrees. Alternatively, the surface may be a l^dimensionai matrix, such as, fer example, a porous scaffold, to ivhleh ceils1 can attach, [0134] in one embodiment the surface modified plate comprises a plate whose surface Contains from at least about 0.5% N, a sum of 0 and N of gmater ilmn or eqpal fe 17,2% and a contact angle of at least about 13.:9 degrees. In an aliemaie emfeodn'nent, the sttrfeee modified plate comprises a plate whose surface contains from at least about 0.5% N, a Sum of O andbf of gr#ter than or ec|utd to 19.5% and a contact angle of at least about 13,9 degrees.

[0135] in one embodiment, the surface tm)dified;piate epmpiises a plate whose surface contains from at least about 1.3% N, a sum off) and N of at least about 24.9% and a contact angle of at least about 20.7 degrees, which is mfered herein as surface modified plate 1, [0136] In one embodiment, the surface modified plate comprises a plate whose surface eon tains from at least about 1,7% H, a sum of O and N of at least about 29.0% and a contact angle ofat leastabont 143 degases, wMefe is refeed herein as surface modified piste 2. 10137] la one embodiment, the surface otodifed ^iafe eomprises a plate whose surface

Contains from at least about 2.0% N. a sum of O and N of at least about 30.7% and a contact angle of at least about! 8.4 degrees, which is refered herein as Mrfaee modified plate 3,

Mone embodiment, the surface modified plate comprises a plate whose surface contains :fem at least about 2,1% N, a sum of O and H of at least : about 30,2% and a eontaef angle of at least about:: 17,4 degrees, which Is refered herein as surface modified. plate 4, |M39| In-One embodiment: the surface modified plate comprises a plate whose surface contains ffptn at least about 1,8% N, a sum of O and N of at least about 28,2% and a eon wet angleof at feast about MM degrees, which is refered herein as surface modified plate 13. 10140] la one embodiment, the surface modified plate comprises a plate whose surface contains fiord at least about 10% %a stun o f O and M of at least about 22, S% and a contac t angle of at least about 443 degrees, which, is sold under the trade name CELLBIND. |0141] In one embodiment, the surface modified plate comprises a plate whose surface contains from at least about 10,2% M5 a sum of O and N of at least about 23,0% and a contact angle of at least about 39.5 ddgreess which is sold under the trade name PRIM ARIA. CItarmMeiixaimn of the Smfkm Modified Elites |0142] in one embodiment, the sfernental composition: of the surface of the surface modified plates may he analysed by K»Ray Ehetoeleetton Spectroscopy (XPS). XPS, also known as Electron Spectroscopy for Chemical Analysis (ESCA), is used ay a method to determine what elements or atoms are present in the surface of a solid substrate (all elements in eoheentrations less than 0,1 atomic pemeat can be detected, except hydrogen and helium:], and to detertnine the bonding environment of such elements or atoms. .As an example, aii XFS analysts of a poiystym«e (e<H3iali® »i3ljf cai^oB an.d hydrogen) solid sample would typically gi ve greater than. 97% carbon, less than 3% oxygen, and 0% nitrogen, ^hydrogen is mpt; detected; different levels of oxygen may be detected due to oxidation of the polystyrene chains at the surface, for example, ante, result of sterilisation by Irradiation): jprevig et o/>, Bioutatarials: 26:3039-3053, 2005; Shea and Horbett, 1. Biomed. Mater; Res. 57:336--345, 2001). 10143) In one embodimetU, the roughness of the surface of tire surface modified plates may be analysed: % Atomic: Pofop Microscopy (AFM), Surtee: atoms or molecules with' a: lateral resolution down;to 1A and a vertical ..resolution: down to 0.1 A can be imaged by AFM.

[0144] in one embodiment the wettability of te;surtee of the surface modified plates may be analyzed by measuring the contact angle. For example, contact angle measurement by the static sessile drop method provides information on foe interaction between foe surface of a sbiid substrate and:a liquid, The contact: angle describes the shape of a Ifouiddfop resting on foe surface of the solid substrate,; and is the; angle of contact of the liquid on the surface of the solid substrate,, measured within the liquid at the contact line wberejiiquidy solid, and gad meet. A. surface with a water contact angle larger than 90° is temied hydrophobic, and a surface with water contact angle less than 90° is termed hydrophilic. On extremely hydrophilic surfaces, that is, surfaces that have a high affinity for water, a water droplet will completely spread (an effective contact angle of OM.

[83 451 In one embodiment, the negative charge d etteity of the surface of the surface nfodffied plates may be analyzed by. measuring the reactivity of the surface with crystal violet. Crystal violet carries a, positive charge, which enables it to bind ionegattvely charged molec ales and parts of molee dies, for example, negative I y charged function al groups present on a polymer suttee. A surface with a high crystal violet reactivity has a higher density of negative charges than a surface with a fow crystal violet reactivity, given that, the surfaces have the same roughness and thus area.

Plot! potent Stem Cells

Ch&amp;mctematsm (^'Phripment Siem C&amp;lk

Muri potent stenreells may express: one or more of the stage-specific embryonic antigens (SSEA) 3 and 4,and markers ..detectable ..using antibodies designated Ira~J~ 60 amiTm-l-Si (XfeomSbri et a/^.:Seibt«s6/2Blil,|45 1.998). Differentiation of plnripotent sfemeells in vitro results In the loss of SSE A~4, Tra-: 1 -60, and. Tra-1 -B1 expression (if present) and increased expression of SSEA-1 . Blndiifcreififeted pluripotent stent ceils typically have alkaline phosphatase activity, wbiehpau be detected fey fbdrig the ceils: with 4¾ paraformaldehyde and.· then developing:: with Vedior Red as a substrate, as described by the manufacturer (Vector Laboratories, Burlingame Calif). &amp;diffe^atiated;plaBt|K.d^t:a®«n..eel.{a also typically express Oet~4 and TERX. as detected by RX-P€it 10147) Another desirable phenotype of propagated pluripoieRt stem ceils is a potential to differentiate into cells of all three germinal layers; endoderm, mesoderm, and ectoderm tissues, Phrripoiency of stem ceils cau fee conErmed, lor example, by injecting cells into severe combined imrmmodeEdcnt (SOD) mice, fixing the teratomas that form using 41¾ parafomtaldehyde: and then examining them histologically for evidence of cell types from the three germ layers: Alternatively, pluripoieney may be determined fey the creadou of emhryoid bodies and assessing the emhryotd bodies ifer the presence of markers associated with the three germinal layers, f 01481 Efopagated pluripotent stem. cell lines: may be karyotyped using; a standard 6-banding technique and compared to published: karyotypes of the corresponding primate species:. It is desirable to obtain eells that: have a "normal karyotype," -which Ricans that the pells are euploid, wherein ah humanchromosomes are present and not noticeably altered.

Saun^^'ofFhuipqimt Stem Cells j'0'149] the types ofpiuripotent stem ceSlsdiiat may be used include established lines of plnripotent cells derived, from tissue 'formed after gestation, ineldding pre-embryonle tissue (such:: as, for example, a blastocyst), embryonic tissue, or fetal tissue taken: any time during gbstation, typically but not necessarily before approximately: 10-1:2 weeks gestation, Non-limiting examples,are established lines of human ES cells or human embryonic germ cells, such as, for example the human ES ceil lines 141,447, and H9 (WiCell). Also contemplated is use of the compositions of this disclosure during the initial establishment or stabilization of,such cells, in vvhieh. case the source cells would be primary piuripotent cells taken dn'ecily from the source tissues. Also suitable ate cells tafesfeom. a piuripotent stem eel! papulation already cultured in the absence of feeder cells., as well as a piuripotent stem cell population already pulfored in. the presence of feeder cells, Also suitable are mutaui human ES cell lines, such as, for example, BOO I v (BresaGen, Adieus, CIA}* Also suitable are cells derived Bom adult human, somatic cells, such as. for examples, cells disclosed in Takahaptt ei oh Ceil 111: 142 <2009% |01SOj In one embodiment, human ES ceils are prepared as described by Thomson e/ a!. (US. Pat. m. [SMiJM};Sewme2S2:n43,1998; Gurr. Top, Dew Biol. 38:133 if., 1998; Prop, Mall. Acad Sci. U.S.A. 92:7844, 1995):,

Culture Piuripotent Stem Celts fill Si 1 In one embodiment, piuripotent stem cells are cultured on a. layer of feeder cells or extracellular matrix protein that support the pluripdtent stem cells in various ways, prior to culturing aceording to the methods of the present in vettrion. for example, phiripofent stem cells am eultumd OP a feeder deli layer that supports pfoliferatiott of plan potent stem ceils without undergoing substantial differentiation. The growth of piuripotent stem cells on a feeder cell layer without differentk^pal^ ^ti^ortM; using (if Obtaining a culture vessel containing a feeder cell layer; and (tif a mediurn conditioned by culturing previously with another cell type, or a ootwcoaditioosd medium, for example, free of serum dr even chemically defined. |d15.2j in. another example, piuripotent them cells are cultured m a culture system that is essentially free of feeder ceils, hut nonetheless supports proliferation of piuripotent stem, cells without undergoing substantial differentiation. The gro wth, of piuripotent stem cells in feeder-cell free culture without differentiation, is supported using (1) an adiayer on a solid substrate surface with one or more extracellular matrix promins; and (ii) a medinm eondiitoned by culturing previously with another cell type, or a non-conditiohed medium, for example, free of serum or even ehemi call ydefitfed. 11153] Is;» alternate embodiment, phmpoteat stem ..cells are cultured:· on a surface modified plate cdnmkiag from at least about 0.5% N|:a sum of 0 and N of greater than or equal to 17.2% and a contact angl e of at least about 1319 degrees in a tnedi um eohetiti Ohsd by culturing previously with another cell type, or a non-condhloned medium; for example, fi-ec of scrum or even chemically defined, ||1S4] Ciiitummeiium: An example of cell culture medium suitable for use in the present invention may be foundib US2002O072I f|. Another example of cell culture medium settable lot use in the present invention may be fonnd in US6642045, Another example of cell culture medium suitable for use in: the present invention may be found In :W:02095014799, Another example of cell culture medium. suitable for use In the present invention may he found in Xu at ul 0|tom Cells 22: 972-980,2004}, Another example of celt culture medium be found in 1)52007001001.1. Another example of cell culture medium suitable for use in the present invention may fee found in Cheon et ai. (BioReprod DOf t l 0,1095ibioireprodJ 05 J46&amp;70; 19 Oct 2005). Another example of cell culture ntedmm suitable for use in the present invention may be found in Leyensieinet ai (Stern Cells 24: 568-574, 2006). Another example of ceil culture medium suitable for use in: the present:hwetsllon may he bund, in US20050! 48070. Another example of eel! Culture ntedmm suitable for useih the present in vention may be found in 125200502:33446. Another example of cell culture m edium suitable for use In the present invention may be found in US6800480. Another example of cell culture medium suitable for use in the presen t invention may he found in 11520:050244962:. Another exampie.of cell culture medium suitable for use in the present invention may he found in 92(32005065354, Another example of ceil culture .medi um suitable for use in: the present invention may fee found in WO2003086845, 10155] Suitable eulfore media may also he ma.de from, the:following components, such as, for example, Duibeeeo's modlftedEagleX medium fl^MBM}, Gibeo # 11965-092; Rnoclout Duifeeceo's modified Eagle-a:medium.(KO PMEM), Oibcp # 10829-018; Hatn-s F I 2/50% DMEM basal medium; 200 mM L-glutamme, Gibco # 15039-027; non-essential amino acid solution, Gibco 1.11.40-050; P^ihercapfoethanoL Sigma # M7522; human recombinant basic fibroblast growth factor (61701¾ Gibco # 13236-029,: differentiation f>fPlurqx)ie0 Slem Cells 18156) la one embodiment of the present inventions pl.ur%otent stem Cells are propagated in eult«res while maintaitrlng their plurtpoiency. Changes in phtnpoieney of the cells with time can he deteratmed by dejecting changes in the levels of expression of mutters associated with phnipoiency, .Mt#^a$ivdly,,ch|hg^ tit piufipptcncy can he monitored by detecting changes in the.· levels of expression of markers associated with differentiation or markers associated with another cell type. 16157] in an alternate embodiment, pi uri potent stem cells are propagated in culture and then treated in a manner that promotes their differentiation into another cell type. The other cell type may he a cell expressing markers ebnractedstic dfthc definitive endoderm lineage. Alternatively, thecell type may be a cell expressing markers characteristic of the pancreatic ehdoderrn lineage. Aiternatlveiy , the cell type may he a cell expressing markers ehameterisfic offhe panereaie endocrine lineage. Alternativelyt the cell ty^:'hmy^:md«fii^pmssi«g;'»mFkera characteristic of the fk cell lineage. |6I5§] Mutipoteni stem cells treated ittaccordanee with the methods of the present invention may he differentiated. into d variety: of:oilier cell types by any suitable method in the art. 16156] For example, pluripotent stern cells treated in accordance with the methods of the present invention may be diiYereniiated tote neural cells, cardiac cells, hepatoeytes., and the like. fdldOJ For example, ρΐηηροΐοηί stem cells treated in accordance with the methods of the present: invention may he differentiated into neural progenitors and cardiomyoeytes according to ihemefhod> disclosed: in. WO:20O7036h7b· |61hl 1 in. another example, pluripotent Mem cells treated in accordance with the methods of the present invention may be differentiated into hcpatocytes according to dte methods disclosed in U'Sipatept f 6162 J For example, pluripoieni stem cei ls may be differentiated into cells expressing markers cbaracteristte of the definitive endoderm lineage according to the methods disclosed in D’Amonrm 0/., Nature BioleehrfoL 23:1534-1541,. 2005. f 6! 63] For example, pkripotent stem cells may be difierendated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed: in Shmozaki mof, Development 131:1051-1002,20(14. 16104] For example, piuripoieot stem cells may be ditforetniaied Into cells expressing markers characteristic of the definitive endoderm lineage aceordimg to the mefbods disclosed in McLean eial, Stem Gells 25:29-38, 2007. |6105] For example, pluripotent stem cells may be difforemiated into cells expressing markers characteristic of tire definiti ve endoderm lineage aecordmgto the methods disclosed mEFAmour v/ al.s Naune Bfoteehuol. 24:1392-1401,2006. 16166] Markers characteristic of the definitive endoderm lineage are selected, from the group consisting of SOX 17, GA7A4, Tlnfolbeia, CSC t crL Nodal, PGF-8, Bmehyury, Mix-like homeohox protein, FGF-4 CD48, eomesodetmiafFOMES), DEN4, FGF-17:,. G&amp;1A6, CXGR4, G~Klt, 0099. and. QTX2, Suitable for use in the present Invention is a cell that expresses at ie#t ope of the markers characteristic of the definitive endoderm lineage. In one aspect of the present invention, a cell expressing markets characteristic of -ip:i^prp|:it.h.0age is a primitive streak precursor cell. In an alternate aspect, a cell expressing markers charaeteristic of the definitive endoderm lineageis:amemkoderm eelL In an alternate aspect, a cell expressing markers ehameteristie ofthe definidye endodem'} lineage is a definitive endoderm cell. 16167] For example, pluripotent stem: cells rhay be difforentiated into colls expressing markers charaeteristic of the pancreatic endoderm lineage according to the methods diselosed in D’Amoor ef nl., Nature Biotechnoh 24:1392-1401,20()6, fOMS] Markers characteristic of dm pancreatic endoderm liueage: are selected from the group eonsistmg ofFdxl, HMF~!beia, PTFla, HKF-6,11139 and PROXL Suitable for use in the present invention is a cell that expresses at least one of the markem chapicteristic of the pancreatic endoderm. lineage. In one aspect of the present; invention, a cell expressing markers characteristic of the pancreatic endoderm lineage is a pancreatic endoderm cell.

[0169] Pluripotent stem cells may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage by any method in the art.

[0170] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage according to the methods disclosed in D’Amour et al., Nature Biotechnol. 24:1392-1401, 2006.

[0171] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by the methods disclosed in D’Amour et al., Nature Biotechnol. 24:1392-1401, 2006.

[0172] Markers characteristic of the pancreatic endocrine lineage are selected from the group consisting of NGN-3, NeuroD, Islet-1, Pdx-1, NKX6.1, Pax-4, and PTF-1 alpha. In one embodiment, a pancreatic endocrine cell is capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endocrine lineage. In one aspect of the present invention, a cell expressing markers characteristic of the pancreatic endocrine lineage is a pancreatic endocrine cell. The pancreatic endocrine cell may be a pancreatic hormone-expressing cell. Alternatively, the pancreatic endocrine cell may be a pancreatic hormone-secreting cell.

[0173] In one aspect of the present invention, the pancreatic endocrine cell is a cell expressing markers characteristic of the β cell lineage. A cell expressing markers characteristic of the β cell lineage expresses Pdxl and at least one of the following transcription factors: NGN-3, Nkx2.2, Nkx6.1, NeuroD, Isl-1, HNF-3 beta, MAFA, Pax4, and Pax6. In one aspect of the present invention, a cell expressing markers characteristic of the β cell lineage is a β cell.

[0174] The present invention is further illustrated, but not limited by, the following examples.

EXAMPLES

Example 1

Passage and Maintenance of II mean Embryonic Stem Cells as Cel Clusters

11175) The human. ES ceil lines HI and H9 were initially maintained: on mitoinyeinC SMciivated. primary mouse embryonic fibroblasts (MEF), The human ES cells· were •y«v ,< .... switchedfront MBS? feeders to Matrigef v' (Beeton-Diefcinson, BedfhrdvyA) over repeated passages; [01761 Treatment of surfaces with Mamge!i vk Growth- Factor Reduced Mafrigel’M was thawed at 4*C and then diluted 1:30 in. cold DMEM/F12' (Invitmgen,. CarGbad, CA), Volumes sufficient to cover the surface Were added to each 6~cm dish (2 ml) or each: well ofa h-well plate (Fmi), :and incubated I hr at room temp. Treated surfaces were used within a: few hours or stored at A-G up to two weeks, [11177] Human ES cell culture: LlndifTeaemkted human ES ceil Colonies (ftpmpitherfhe 119 or HI· lines) were harvested: from feeder layers by incubation in I mgripfcollagenase l¥ (Sigtha-Aldrich, :St, Lotus, MQ) bv:DMEM/P12 lor W tninutes./fpllpwed by scraping with a pipette. Cell clumps were pelleted by centrifugation. at 600 x g for four minutes and the pellet dispersed gently with, a 2-mi pipette to break colonies into small clusters of cells. These eell clusters were seeded onto Matrigei* ^-treated dishes in media conditioned with rmmse emhryonfe dbroblasts (MEF-CM), further supplemented with bFGF .(8 ngdnh H&amp;D Sysietu%vyhme^6l%''MMb at 5()-150 colonies per 6-em dish in 5 ml growth medium, Medium was changeddatly.

Colonies on MairigeliM in. MEF-CM became large and were passed when they occupied 70-80% of the surface area, approximately every 3-4 days. The: human :ES cells In the colonics had a high nucleus to cytoplasm ratio and had prominent nucleoli similar to human ES cells maintained on feeders (Figtue 1), Dirfbrettiiated colls ropresented less than 5¾¾ of total colls in. etdiure.

[0178] For Toutine passage of cells: in 'MEF-GM on Matrigel1JVis cells were incubated. in I mgl'ml eollagonasoTV ip..DMB;M/F.12 fbrup to hOminules andremowed from the dishos by fbrcelbl streams of DMBM/P12 with scraping. Cells were pelleted, dispersed, and seeded at a 1:3 or 1 ;4 ratio.

Example 2

Passage of Human Embryonic Stem Cells as Single Cells [0179] Human ES cells of the cell line H9 were grown as single cells according to the methods disclosed in US Patent Application LFS5163USPSP, assigned to LifeScan Inc. Cells were passaged by treatment with TrypLE™ Express for five minutes at 37oC, and seeded at 10,000 cells/cm2 substrate surface.

Example 3

Attachment, Cultivation and Maintenance of Pluripotency of Human Embryonic Stem Cells Using Surface Modified Plates Lacking Extracellular Matrix Protein/Components and Feeder Cells [0180] Human ES cells of the line HI, at passage 49 were maintained in MEF conditioned media on Nunclon DeltaTM plates treated with a 1:30 dilution of growth factor reduced MatrigelTM, prior to study. Cells were dissociated from the surface for passage by lmg/ml collagenase dissociation or by manual scraping.

[0181] These cells were then seeded onto two untreated wells of the surface modified plates (6-well format). Additionally, one well of each plate was treated with 0.1% xeno-free human gelatin as a control. Cells were also plated directly onto untreated and gelatin-treated wells of CostarTM (cat. no. 3516; Coming, Coming, NY), FalconTM (cat. no. 351146; Becton Dickinson, Franklin Lakes, NJ) and Nunclon DeltaTM (cat. no. 140675; Thermo Fisher Scientific, Roskilde, Denmark) 6-well plates for negative controls, and plated onto wells treated with 1:30 dilution of growth factor reduced MatrigelTM to provide as positive controls. In all treatments cells were maintained in MEF conditioned media.

[0182] After two passages, surface modified plates 2, 3, and 4 had attached ES cell colonies, which re-attached to the plates and grew following enzymatic dissociation. There was no apparent difference in rate of attachment or growth in gelatin or untreated wells from surface modified plates 2, 3, or 4.

[0183] Cells mechanically dissociated from plates treated with 1:30 dilution of growth factor reduced MatrigelTM were poorly attached to surface modified plates 2, 3, and 4, while_ cells enzymatically dissociated with I m^%icftliagcaasc/:w^e\weliatiachadi.a gelatin or untreated wells froffi surface Modified, plates 2? or 4. I'8184J I‘ilp49 ES eells added to surface ntodtfied plates 1 artdd-12 w&amp; to tmtreated or |οΜ»:'ίίθ&amp;^Ν\ι.«:δ:Ιο«.::ΡεΙί&amp;'?·Μ plates, Falcon m plates, and:Costar!M plates did hot attach. Theissme cells did. attach to plates treated with 1:30 dilution of growth factor redae^dlMstrigel · ^ indteatingdhat. th&amp;ce!J&amp;,wsre. compedeir&amp;jo attach to a substrate surface. f8l85] Normal passage time for ES cel ls of the HI line plated on MO dilution of growth factor reduced Matrigei kM was 3-4 days, however cells plated on surface modified plates 2,3and 4 took 7 days of culturing before they were ready fef passage. This wMpmbabiy due to the reduced rate of attachment on the treated surfaces, since more starting colonies were apparent on Matrige!m-treated surfaces hnniedialciy after plating than on Surfaces 2, 3 and 4. 18186] the passage (p) 58 Cells were split at a 1 to 2 ratio and half of the sa mple was collected for ENA purification and tested ^'ex^^toa oC^luripotmcy markers (Table 1). The other half of each sample was replated to surface modified plates. Colonies thatdpltned at this passage (pS lTaSM pqtured 7 days of culturing before they were ready to he passaged, apd.tb&amp;ismaJl .oobrues- that dpyebpd after only 4 days of enduring are shown in Figure L These colonies ntainiameei Classical ES cell colony morphology. |8187] Cnlturcs were stopped at passage 4 on surface modified plates 2, 3 and 4 and samples were assayoddor plorijmfeney mafckdhs'by 9RT-PCRs(Td6^:-2) and diflereniiated to a definitive endoderrn fate (HE). Cells at passage 4 maintained expression of the classical piufipptency mH^ers: 0et4, Nanog, Sox2, and ΤΕΕΊ', Ihnthermore, the cells were able to differentiate to a definitive endoderrn fate upon exposure to a media eomainipg DMEM/F12,1 Ofegdnl Activist A, 20 ngdnl Wntda, and lhS-2.0% EBS (Table 3) indtcidtug that ploripotemty was 'kdi^imed in the cells through passage d.

Example 4

Attachment^Cnlfiv^tlo» and Maisitenanee of Piuflpoiency of Homan Embryonic Stem Ceils on Sorfaee Modified Plates Lacking Extracellular Matrix

Ifootelii/tloiBpeBeRts and Feeder Cells: Effects of Rho lohlfettfeo and Rfe©

If isase inhtbltlo» |0I88] Homan FS cells of the Mne HI, at passage49 were maintained in MEF conditioned media on Nonclon Doita1 opiates treated with a 1:30 dilution of growth factor reduced MatrigdP1, prior to study. Cells wcte:$s^iaied surface for passage by

Img'ml collagenase dissociation. 10189} Thesecells ware then seeded onto untreated wells of surface podified plates: p~weli formaf)> Cells were also plated directly onto untreated and:gelatin-treated wells of COstar^5 FalcW'^l -fed Nunclon DeIta{M fowelt plates for negative controls and plafod phto we!Is treated with 1:30 dilution of growth fectof reduced Marrig^ii;M id-pro vide: as positive: controls, in all treatments cells wore maintained in MEF conditioned media. 101.90) Human. ES cells of the line HI, at passage 49;a0dte4.:io.St^^e::-)WCEiificdi:pis.l^ 1 and 5-12 and to untfoatedfor gelatin treated Nunclon Deltai M plates and Cdstar1M plates did not attach, however, they did attach tosurface modified plates,¾ 3, and 4. The same cells did attach to plates treated with 1:30 dilution of gmwfo foetor reduced Mdtrigel indicating font the cells were competeht to attach to a substrate snriaee, [0191] Normal passage time for Η1 ES ceils plated on 1:30 dilution of growth factor reduced MatrigeliM wns 3-4 days, however cells plated oh surface modified plat#: 2, 3 and 4 took 7 days of culturing before they were ready for passage. This was probably due to the reduced rate of attachment on. the surface modified plates, since more starting colonies were apparent on jViatrigel 3&amp;5-treated surfaces immediately after plating than on. surfoee,modified plates 2, 3 and 4. |0192:| The passage :(p)iO Cells were split at a 1. to 2 ratio and halfof tlfosafople was collected for ENA purification and tested for expression of pluripotency markers (Table 1), The other halfof cads sample was replatcd to surface: modified plates, Colonics that fopfod at: this; passage (p51) also required 7 days of culturing before they were ready to he passaged, and. the small colonics that: developed: after only 4 days of culturing are sho wn in Figure L "These colonies maintained classical ES cell colony morphology.

[0193] Due to the delay in passage, the cells were split at a 1 to 2 ratio and half of the passage 4 samples were plated in MEF conditioned media or MEF conditioned media supplemented with the Rho kinase (ROCK) inhibitor, Y-27632, at a 10 μΜ concentration in an attempt to improve cell growth kinetics. Cells were kept in the plating media for 48 hours after passage at which time the media was changed to fresh unsupplemented MEF conditioned media.

[0194] The addition of Y-27632 at a ΙΟμΜ concentration significantly increased plating efficiency of the cells (p52) and the improvement in colony growth was apparent after 4 days post-plating (Figure 2). Alternatively, prior to collagenase dissociation, human ES cells of the line HI were also treated with 0.5 ng/ml of a cell permeable form of the Rho inhibitor, C3 exotransferase, which also increased the plating efficiency of the cells.

[0195] While cells plated in 10 μΜ Y-27632 could be passaged 4 days after plating, cells plated without the ROCK inhibitor were not ready to be split 4 days after plating. Cells treated with Rho inhibitor, C3 exotransferase were also not ready for passage 4 days after plating and cells exhibited increased differentiation to a fibroblast-like morphology. Consequently, cells treated with Rho inhibitor at passage 4 were treated with Y-27632 at all subsequent passages (Figure 3).

[0196] Cells were further passaged to at least 10 passages on surface modified plates 3 and 4 and were tested for the presence of markers associated with pluripotency: genes by qRT-PCR; cell surface marker expression by flow cytometry; and immunofluorescence of cell surface and nuclear proteins (Figures 4-6). Cellular pluripotency was also confirmed by testing their capacity to differentiate to definitive endoderm, pancreatic endoderm, and form embryoid bodies composed of the three germ layers (Figures 7-9). Cells were also tested for karyotypic stability, and we observed that cells could maintain a normal karyotype (Figure 10).

Example 5

Attachment and Detachment of Human Embryonic Stem Cells Through Rho Kinase

Inhibition [0197] Human ES cells of the line H9, at passage 40 were maintained in MEF conditioned media on Nunclon DeltaTM plates treated with a 1:30 dilution of growth factor reduced MatrigelTM, prior to study. Cells were dissociated from the surface for passage by 1 mg/ml collagenase dissociation or by manual scraping.

[0198] These cells were then seeded onto surface modified plates 2, 3, 4 and 13 (12-well format) in the presence of increasing amounts one of the following Rho Kinase inhibitors: Y-27632 (from Sigma, St. Louis, MO or EMD, San Diego, CA), Fasudil (Sigma), or Hydroxyfasudil (Sigma), and maintained for 3 days, each day replacing the media and compound. At the end of day three, media was removed and the plates were stained with Crystal Violet (0.5% in water) to visualize colonies.

[0199] By day three, surface modified plates 2, 3, 4 and 13 had attached ES cell colonies in the presence of increasing amounts of Rho kinase inhibitor. Best results were obtained through the use of Y-27632 (10 μΜ), although some colonies could be observed to attach and grow with the Rho kinase inhibitors, Fasudil and Hydroxyfasudil (Figure 11).

[0200] It was attempted to determine the optimal dose of Y-27632 to promote cell binding, by treating cells with a range of plating concentrations of Y-27632 for the first day of culture. After the first day in culture cells were treated on subsequent days with a 10 μΜ concentration of Y-27632. We observed that the maximal concentration to stimulate attachment and growth of ES cells was 10 μΜ (Figure 12) and that this occurred on surface modified plates 2, 3, 4, 13 and CellBINDTM (Corning, Coming, NY).

[0201] The effect of treating the cells continuously with a single dose of Y-27632 on attachment and growth was also tested. The cells were dosed with 0, 1, 4, or 10 μΜ Y-27632 for 4 days. Some binding was observed on surface modified plates without treatment (0 μΜ), however the optimal concentration to stimulate attachment and growth of ES cells was 10 μΜ Y-27632 (Table 4) on surface modified plates 2, 3, 4, 13.

[0202] Since the addition of ROCK inhibitor significantly enhances the plating and growth kinetics on surface modified plates 2, 3, and 4 versus untreated cells (Figure 13), we wished to determine if this was due to maintenance of proper cell attachment or due to increased cell proliferation. It was observed that Rho Kinase inhibition does not increase cell proliferation, because cells treated with Y-27632 grow at a similar density as untreated cells (Figure 14). Instead, Y-27632 treatment maintains the attachment of cells to the surface and allows them to grow with normal proliferation kinetics (Figure 15). Removal of a Rho Kinase inhibitor from the growth media of ES cells plated in the presence of Rho kinase inhibitor results in detachment of the cells from the surface. The formation of embryoid bodies with differentiation to the 3 germ lineages is accomplished by culturing ES cells in a suspension.

Consequently, although later reapplication of a Rho kinase inhibitor restored attachment of cells (Figure 15), as expected, substantial differentiation of the ES cell culture was observed in samples where Rho kinase inhibitor was withdrawn for 24 hours of culture and cells were allowed to detach, grow in suspension for 24 hours and Rho kinase inhibitor was then reapplied.

Example 6 H9 human ES cells passaged with TrypLE™ Express on Surface Modified Plates Show

Improved Adhesion with Y-27632 [0203] Initial passaging of H9 human ES cells onto surface modified plates. Adhesion is improved with continuous 10 μΜ of Y-27632. This is true for the four surface modified plates tested: 2, 3, 4 and 13. Images of H9 cells 24 hours after seeding on Surface 3 are shown in Figure 16.

Example 7 H9 single Human Embryonic Stem Cells Passaged with TrypLE™ Express on Surface

Modified Plates Remain Pluripotent [0204] Human ES cells are pluripotent and have the ability to differentiate into all cell lineages. The pluripotent state of the cells must be maintained by the surface on which they grow. To determine if the surface modified plates can maintain human ES cell pluripotency, the human ES cells were passaged 38 times with collagenase and 38 times with TrypLE™ Express followed by 5 passages on surface modified plate 3 (Surface 3), surface modified plate 4 (Surface 4) or MatrigelTM at 1:30 dilution. 10_ μ M the media of indicated samples. The expression, of pluripotency markem Tra-l-bO, : Im-i-Bl;, SSEA-3 and SSBA~4? was evaluated by flow cytonrotty. iE^sulM#stfown do Figure 17. The postage positive ceils Is ind icated on the y-axis, Single human E$ ceils grown or--surface modified plates 3 and 4 can tnaintain their pfonpotency.

Example^

Rho Khrose Inhibition Promotes Adhesion and growth ofCelisTrem the Human Embryonic Stem Cell line 119, Crown as Slagle Cells on Surface Modified Plates

open Transfer from MafrlgelJM f 82dSJ The role of Y-27h32 in human ES cell adhesion and cell growth was studied in relation to the surface modified plates. i!9 human ES cells were passaged 38 times with eoilagenase and 50 times with TrypLETW Express followed % seeding onto Surfaces modified plates 3 or 4 (naive cells). ..Alternatively B9 human ES ceils passaged 38 times with eoilagenase and 38 times with Triple Express followed by 9 passages on surface modified piate 3 (Surface 3, acclimated ceils), or surface modified plate 4 (Surface 4, acclimated cells). Cells were: seeded at a density of 1O'VcnF in MEF conditioned foedia andgrowii for; two days with or wdthput the presence of 10 p.M :ofY47|32. Results aroslmwn in Figure 18, ¥-27632 improves attachment of narye cells to surface modified plates 3 and 4. ¥-27632 dld not Improve aitaohment of acclimated:;ceils to surface modified plates 3 or 4. surface modified' plate 3 Improved attachment and/or growth of naive cells. Surface: modified plate d imprpyed attaGhmesrt sodfor growth of acellrnaied human ES cells, Idle cells were followedibr a total of 4 days (Figures: 19 and 20). The naive single cells exhibited an increase growth rate when, cultured with ID μΜ Y-27632 with Surface modifledpiate 3 showing a slight advantage (Figure 19). The acclimated single cel ls exhibited improved growth rotes with out the 10 [tM of ¥-27632 (Figure 20).

Example 9

Surface Modified Flutes can be used to Screen Cumperotds fbSCIbj Surface: modified, plates In Od-wed configuration and. in the Society for Blomolecular

Screening :(SBS) standard format can be used for growing single: htsman iES ceils in. the pmseace of 10 μΜ Y-27632. Images of H9 single ceils plated in ftp-well plate wells are shown is Figure 21, this would allow for the screening of compounds direetiy m ^weil plates with so Interfering cel ls or adiayers, such as mouse emhrvonie fthreblasts or Matrlgei'M, respectively,

Single Embryonie St«i»-Cells Cultured, on Surface ModifiedPlates are able to Differentiate Into Definitive Emituferm |b2|7) One goal is to differentiate human BS cells into dtflerenteeli lineages, to: determine: if surface modified plates: can support diftfeshtiafibn, bamanlS cells were passaged 38 tipes with eollagenase and 38 times with TrypLETM Express followed by 9 passages on surface modified 'plate·3 (Surface 3), or surface modified plate 4 (Surface 4), Aisapoatiye eoptoh human FS ceils wem gmwn on MairfgelJ>4 at 1:3$ dilution, 10 gM of ¥-22032 was added to the media during expansionof ludlcaied cell samples, After cell expansion, the ability of the cultured cells to form definitive ehdodtfm.wM-.'fevdli^eti, IMefly, 703¾ confluent cultures were treated with 100 ng/mi Activin A, 10 ng/mi Whtda and 0.5%¾ FBS k· DMBM-Fii.inedia f&amp;Ytwo days, 1 he treatment was followed by 3 days with 100 ng/m! Activin Λ and 2%FBS in DMESlrf7l2, Cells diffimemiated into definitive endoderm am identified by CXCR4 protein: expression, via flow cytometry (Figure 22). fbe percentage of positive cells: is indicated on the y-axis, FIutnan BS cells cui tnred: as single ceils Can differentiate into definitive endoderm in the presence or absence of Y-27632 on surface: modified plates 3: and: 4,

Example 11

Single Embryonic Stem Cells Citltured on Surface Modified Plates are able to Differentiate Into Iknereafk Endoderm 10208} After completion'ofthe definitive endoderm pfoioooi, the cells were incubated tot 3 days with FGP-7 (50 ng/ml; R:&amp;p Systems), the sonic hedgehog .inhibitor, EAAD eyetopamine (2.5 μΜ; Sigma^Aldrich) and 2%¾ FBS in DMEM-F12 medium, At this point, ceils not treated with Y-27632 during expansion detached Irani the surface modified:plates 3 and 4, The cells tmaied with Y-27d32 during expansion, were incubated m additional four days with FGF~? (50 ng.'ml h KAAD cyclopanrine (2.S $1¾ Retinoic Add (l Sigma-Akin oh) and 1% Be 7 flnvitrogen) in DMEM-F12 (posterior fbrcgiit stage*;FF}:, After this umc, cells were incubated as additional four daysan Exeudiri 4 (50 (1 pMf Calhiochem),and 1% B27 in ΏΜΕΥΙ-ΕΙ 2. Diifeteittiadod was eo.nt.in ned to foe pancreatic endoderin stage (EH), This entailed a fhtee»day ireahuent with. CMRL medium (Invitmgen) containingSOngfoi!, HGF, IGF (RltD Systems), and Exendin 4 (SOng/ml), and 1% B27, RNA .iitnples were taken at stages FE and EH from one well oi the surface modified plates 3 and 4. These samples were then analyzed: by real-time PCRat this step tor pancreatic markers IF#!* Nkx2,2, Pax4, NeurolA HNF3b, Plfla, insulin and AFP.. Evaluation of the same pancreatic endoderm markers was repeated at this stage, RHA samples Iron! untreated h uman ES cells of the same line were subjected to real-time PCR in parallel to treated satnpfes. Treated samples were normalized to untreated controls set to a fold change of 1, Pdxl and insulin, expression was monitored and compared between surface modified plates, 10209) Induction, of pancreatic endoderm markers wasobsetved from ceils treated on surface modified plates 3 and 4, although expression was: higher with cells treated bn surface modified plate 3: (Figure 23), Both; surface modifiediplates in the presence of Y~ 27632: during expansion can support the differentiation of single human. ES ceils to posterior foregui and pancreatic endoderm. whereas single ceils not treated with. Y-27632 during expansion detached prior to posterior foregut differentiation.

Example 12 fit and S9 Human ES Ceils Adhere· to Surface Modified Plates and Adherence is Enhanced in Treating Cells Alth Y-27632 {02.1.0) Passage 49 114 humanES cells previously plaied fo: 1:30 Matrigel1^ treated plasti c ware andlpsWtt. :k MEF.eondinoned media supplemented with Bng/ml of bFGF were LIBERASE treated and plated fo surfacemodified plates in. MEF conditioned: media supplemented witbRng/ml of bPCiF and not otherwise treated or suFpfetnented. We observed, that 24 and 4F hours after plating 1|0 human ES cells: to surface modified plates small, colonies, could be observed on: Surfaces 2-4 and 13, and CellBIND1*'·, and PrimariaiM (eat, no. 353846, Becton Dickinson, Franklin Lakes, NJ) with crystal violet stain (Figures 24-26). Furthermore, the adherence of H9 human ES cell colonies was improved by the addition of Y-27632 and the effect was dose responsive (Figure 25). Low concentrations of Y-27632 (1 to 2 micromolar) showed a minimal improvement in human ES cell attachment versus untreated human ES cells (Figure 25) while higher concentrations of Y-27632 (4 to 20 micromolar) promoted adherence of human ES cells to surface modified plates as measured by crystal violet stain (Figure 25 and 26).

[0211] In addition to the dynamic regulation of human ES cell attachment by addition of Y-27632 to the cell culture media, different rates of adhesion of human ES cells to various surface modified plastics in the presence of Y-27632. For example, cells were less adherent to CellBINDTM plates and were more likely, over time, to detach from CellBINDTM plates even in the presence of sustained Y-27632 treatment while cells were more adherent and less likely to detach from surface modified plates, 3, 4, or 13 or PrimariaTM when treated with the Rho kinase inhibitor, Y-27632 (Figure 25 and 26).

Example 13

Cells from the Human Embryonic Stem Cell Lines HI and H9 Attach and Form Colonies at Different Rates on Surface Modified Plates in the Presence of Y-27632 [0212] HI and H9 human ES cells previously plated to 1:30 MatrigelTM-treated plasticware and grown in MEF conditioned media supplemented with 8 ng/ml of bFGF were LIBERASE treated and plated to surface modified plates in MEF conditioned media supplemented with 8 ng/ml of bFGF and not otherwise treated or supplemented with 20 micromolar Y-27632. Forty-eight hours after plating H9 human ES cells to surface modified plates 14 and 15, small colonies were observed when the media was supplemented with 20 micromolar Y-27632 (attachment and colony formation was variable from experiment to experiment) (Figure 27). HI human ES cells also attached to and formed colonies on both Surface 14 and 15 in media supplemented with 20 micromolar Y-27632, and this was more prevalent than the binding observed with H9 human ES cells. These data indicate that there is human ES cell line-to-line variability in attachment to and colony formation on solid substrate surfaces.

Example 14

Human ES Cell Attachment to Surface Modified Plates Using Defined Media [0213] Passage 49 H9 human ES cells were passaged twice in the define media, mTeSRTM, on MatrigelTM-treated plasticware. The cells were then LIB ERASE treated and plated onto the surface modified plate Nunc4 in mTeSRTM media. Cells were either plated in media with or without 20 micromolar Y-27632. Wells were also treated with various proteins for 30 minutes prior to seeding cells (no treatment, 0.1% gelatin, 2% BSA, ,34mg/ml rat Collagen I, 1:1000 MatrigelTM, or 1:5000 MatrigelTM) to determine if these proteins could promote human ES cell adhesion in defined media with or without Y-27632 (Figure 28). In the absence of Y-27632, human ES cells plated onto a surface modified plate in defined media did not attach-even in the presence of extracellular matrix proteins such as Collagen I or 1:1000 MatrigelTM. However, when 20 micromolar Y-27632 was added to define mTeSRTM media, human ES cells adhered to surface Nunc4. Furthermore, this adherence was equivalent in untreated wells and wells treated with 0.1% gelatin, 2% BSA, and 0.34 mg/ml rat Collagen I. There was a modest increase in human ES cell attachment in wells with low concentrations of MatrigelTM (1:1000 and 1:5000 dilutions), however these concentrations of MatrigelTM were insufficient to promote adhesion in the absence of Y-27632. These results demonstrate that in the presence of the ROCK Inhibitor, Y-27632, human ES cells can be cultured on modified plastic substrates in defined media and that low concentrations of MatrigelTM of about 1:1000 or 1:5000 can improve this adhesion.

Example 15

Surface Modified Plates in a Flask Format can Promote Human ES Cell Attachment and Differentiation to Definitive Endoderm and Pancreatic Endoderm [0214] HI and H9 human ES cells previously plated to 1:30 MatrigelTM-treated plasticware and grown in MEF conditioned media supplemented with 8ng/ml of bFGF were LIBERASE treated and plated to T25, T75, T150, and T175 flasks at a 1:2 or 1:3 seeding density onto various size flasks with modified surfaces. The cells were seeded in MEF conditioned media supplemented with 8ng/ml of bFGF and 20 micromolar Y-27632. Human ES cell colonies were then allowed to grow, with daily media changes of MEF conditioned media supplemented with 8ng/ml of bFGF and 20 micromolar Y-27632, until the plates were approximately 50% confluent. At this time the media was changed to DMEM/F12 media containing 2% BSA, lOOng/ml Activin A, 20ng/ml Wnt3a, and 20 micromolar Y-27632 and the cells were maintained in this media for 2 days with daily media changes. On day 3 and 4 the media was changed to DMEM/F12 media containing 2% BSA, lOOng/ml Activin A, and 20 micromolar Y-27632. Cells were then released from the surface with TrypLE and assays by flow cytometry for expression of the definitive endoderm (DE) surface marker, CXCR4. It was observed that under these conditions, human ES cells differentiated to a highly CXCR4 positive population, that was as high as almost 90% CXCR4+, indicating that the cells were mostly differentiated to definitive endoderm (Table 5). Furthermore, the attachment of the cells to the culture surface during growth or during differentiation was dependent on maintaining ROCK inhibition, since withdrawal of Y-27632 from the culture media resulted in cell detachment from the plastic.

[0215] To determine if pancreatic endoderm could be formed from the definitive endoderm derived on surface modified plates in flask format, the cells were incubated for an additional four days with Y-27632 (20 micromolar), FGF-7 (50 ng/ml), KAAD cyclopamine (2.5 micromolar), and 1% B27 (Invitrogen) in DMEM-F12 and then an additional four days in this media supplemented with Retinoic Acid (1 micromolar; Sigma-Aldrich) to differentiate the cells to a pancreatic endoderm stage. RNA samples were then taken and analyzed by real-time PCR for the pancreatic marker Pdxl. Treated samples were normalized to untreated controls set to a fold change of 1. We observed that samples had increased levels of PDX1 versus undifferentiated human ES cells, with mRNA levels at least 256 fold higher in the differentiated cells than that observed in undifferentiated human ES cells.

Example 16

Surface Treatment and Surface Modified Plates i till hj Surihee modified plates were prepared ty? tearing try cotton molded: i terns using a corona plasma treatment or a microwave plasma treatment (Table (>}. The polymer mateoals used in injectiou molding were polystyrene, polycarbonate, a blend of polycarbonate and polystyrene, ami cyclic olefin copolymer. The surface modified plates were Individually packed In plastic bags, then sterilized by gamma iemdiaiion 125 kGyT and finally stored at room temperature until used m cell culture or surface cMaaeteifi^atibjR' e*p®itaeaM.. 'Surface modified plates 1%30 and 31-32 were molded using the saum pulyi^ plates 1% 33 and 34, re^^vdlyfbubw«a®'::flOip:1^^3ift''teaied. Surfaces: 14 and.Hi were not gamma irradiated. pi7] Corona plasma treatment was carried out in a metal vacuum chamber with only one electrode inside the chamber and electrically isolated from the inside of chamber (€~ Lab Plasmay Vetnphone A/S, Denmark). The metal waifs served as counter electrode (ground)- A sel fitualng corona generator generated the electrical field giving sufficient energy to generate plasma in tbe entire chamber. An item to be treaied was placed at the bottom of the chamber. The chamber was closed and evacuated to a pressure of If'2 mbar. At this pressure the valve to the vacuum pump was closed: and the corona generator engaged. The generator was set iq generate an Output of 2000 W. The plasma was energized for: 5 to 60 seconds, The gas inlet valve (air) was then opened, and the pressure in the chamber returned to atmospheric level. {02181 Themicrqwave plasma treatment was carried out in a quarts vacuum, chambers (Model 300-B for surface modified: plates 5-1.2 and Model 440 for surface piedified plates 1.4 and 15; both from Icchnics Plasma GmbH., Germany), The energy to generate the plasma was supplied by a 2,43 GHz microwave generator outside the chamber. An item to fee treated was placed on a gfass plate inside the chamber;. The chamber was closet! and evacuated to a pressure between 0,3 and 0.5 mbar. The valve to the vacuum pump was kept open, and the pressure was maintained at the desired value by adjusting gas (air or oxygen) flow with the: gas inlet valve. The microwave generator: was thou engaged. The generator was set to generate an output of 500 or 600 W, The pump valve was then closed, and the air inlet val ve was opened, in order: to bring the pressure in the chamber to atmospheric level. I Oil 9) Fable 6 shows pow er, time, pressure, and gasses used in preparing surface modified plains by corona plasma or microwave plasma.

Example 1?

Surface Clharaeteflzatlon of the lurlte Modified Plates of the Present Invention

Water Comaci Angies |0229) Surface-modified plates 1:-4 and 13: were mdividaally packed to.plastic bags* sterilized, and stored at r«m temperature foreughdut the test period. Contact angles were fi rst measured one week ate surface treatment and sterilization, and then again at the time points given In Figure 2f, AM contact angle measurements were done using the static sessile drop method and a PCPX measuring Head fiom FIBM) Systems AB, Sweden [goniometer consisting ofvideo camera and computer software (v. 3.1)]. Tfe tangent leading method was used for caleuiatiod of the contact angles. Drops of 4,0 pL MiMiQ water was applied using automatic drop application in static mode, according to' the tpaUu&amp;etures instructions. T he contact angle of each drop was measured once (7 drops were: applied: to each sample per time point). For each time pomp a new sample was used in order to avoid any infiuence from earlier moasurements. Measurements on Nuncfon Delta1 M and. CgS;|BiNiDlM surfeces; was performed under the same experimental conditions as measurements on Surface 1--4 and 13,. but the surffeg treatment and sterilization was dope More than 12 w#hs before the:; first: measurement (Nunclon Delta5 M* was sterilized one week before the; first measuremenf i Figure 29 shows that surface modified: plates 1.-4 and 13 were of similar hydrophilicity and more hydrophilic (lower water contact angles) than. Nonci©U:DeSia;lM smd CelSBfMD*- surfaces. The hydrophilicity of surface modified plates 1-4 andl.3 was stable for af; least 12 weeks;afief sufitce treatment and sterilization. 1922 !) Celi;B!ND^M has pffefonsiy been described as having a contact angle of 1.3 ,4 degrees (standard deviation of 4 degrees) [Corning Technical Report (2905), Coming^ C'ellBlNB® Surface: Ah Improved Snrfaee for Enhanced: Cell Attachment (CLS-AM-057 REV!) on GLS...Mi;0S7paf].

Negative Charge Demiiy f0222) The te^^':efir^^ve-''elkrg^''0n surface modified plates I -4 aad 1:3¾ Nunclon surface, Prjmai4aiM surface. Falcon^and a n0n>tf©8idi ftafc steriMjEedl'poIySfyrcnc surface (all in 3-cto dish format) was determined, Three ml of aqueous crystal violet solution ¢0,015% w/v) was dispensed, in each dish, and dishes were incubated for 60 minutes at mom temperature under gentie shaking (50 rprn). In order to remove crystal violet not bound to the surfaces, the dishes were washed three times with 3 ml MiiiiQ water, and then dried Over night at 60od The crystal violet bound to the surface was desorbed by addition of 1.5 ml of OJ: M BO in BtOH solution {99W and mcubatingths dishes for 2 minutes at room temperature under gentle shaking (50 rpm). Absorbance of the HCfEtOH solution with desorbed erystal violet was measured at 590 am using an Bn Vision 2100 pheroplate reader (Perkin Elmer; Waltham, MA, USA:). Ahsoihance values were corrected for background absorbance of HCEEtOH solution. The negative charge density was measured On three dishes per snrface, and absorbance measurement was performed in triplicate for each dish. 103231 The negative charge density for surface modified plates is shown in Figure 30, The negative charge densities of Surfaces :1>4 and 13 were similar, hut longer surface treatment time in the interval of 5-60 seconds tended to result in a..lower surface negative charge density. Surfaces 1-4 add 1.3 had significantly lower negative charge densities than €eiiBINDiM surface and a Nuncio»OeltalM surface treated in 2002. Surfaces 1-4 and 13 had negative charge densities at the same level as a'Noneidn Deltasurface: treated in 2005, and signiSeanfiy higher negative charge densities than Frimaria1- surface. Falcon1- surface, and a non-treated (but sterflizedl polystyrene surface. The lower negative charge density of Nuncio» Deliai M surface treated in 2005 than of Hunclon DeltaiU surface treated in 2007, suggest that surface-treated polystyrene hecotues slightly less negatively charged over time. The high lev# of negative charge density of GellBiND5^ is not because of higher snr&amp;ee roughness and thus surface area (See AFM analysis in this Example). X-Ruy Phmodeciron Spectroscopy (ATS) |0224j Sut&amp;c^'isi^fed'':plates 1~4 and 13-! 5, atod plates with NundOn Delta1C?ostari"5, Falcon5**, €el!13|::ND'm and Primaria1** mufaees mm. analyzed using XPS. Sample was presented: to the x~ray source fey cutting sections imm. the-plates and niounting them withppring: clips onto a stainless steel sample holder. Samples were mediated with. Aj koradtatioa (I486 eV). lire analysis way perform ed with an angle of 45* between the sample and analyzed the spectra were curve fit using: the software package provided by the insimment’s vendor. Physical Electronics, The software; bilked commercial Mutlabi;vi routines for data processing. The iastfomeni used for the analysis was a Physical Electronics Model JdOCl X-Rav Phoioelecptitt Spectrometer. llo:'Q«tcrmc^':two:.totSwmnombtem.m depth in a region of about one mtllimeterin pad of the ptaies was analyzed in each of two plates per surface. 102251 Surface elemental composition in units of atonne percent Is shown in Table % All surface modified plates contained carbon, oxygen and nitrogen (hydrogen is not detected in XTS| in the surface, Surfaces 1-4, Suffice 13 and Cel1BlNDiM surface contained more oxygen than the other surfaced analyzed, SnrfaCes D4 and Surfaces 13-15 contamed: i:ess: nrtmgen thaB. Primari8iM, but more nitrogen than the surfaces of biuuclon DettaiM, Clostar5^ BalCOn**4, and CeliBINDm plates; Oxygen and: nitrogen levels correlated positively with longer surface treatment time (Surfaces 1-4 and :1.¾ and Pie highest levels of both of these elements were obtained, using 30 or 60 seconds of corona plasma tmaimeni (Surface 3 and Surface 4, respeefi veiy|.: sSnffaees 3 and: 4. were similar in elemental eomposition. Surfaces 2 and 13 were similar in elemental composition, and more like Surfaces 3 and 4 than Surface 1 in elemental composition. {02¾¾ Clx spectra peaks were curve fit (heat chi-squared fit), is order to identify and quantify the bonding etwimnmema fer carbon in the surfaces.; by using peak widths dp'd::dtegyld^tioii|'.fdf species as found in the literkture (Table 8)., The coucentratious am reported in units of atomic percent, which, were: obtained, by multiplying the area percent:by the atomic concentration. Surfaces 2-4 and 13 were similar in terms of the. carbon bonding environments. The proportion of carbon in bonding environment was lower in Surfaces 2-4 and 13 than. In the other surfaces analyzed. The proportion of carbon in O-p^Oj-O bonding erwironment was higher in Surfaces :2-4: and 13 than in the other surfaces analysed. Similarities between Sin-faces 2-4 and 13 and Surface 1, €eMBlMDl^irfhcfo and/or Primaria'y surface: were also identified. The proportion of carbon in C-O-C or C~ bili3Ai"bondtng eavdionmoai^me energyfoeationfo specrfa) was higher in Surfaces 1-4 and 13 than in the other strffoees: analyzed,' Tlg^portkp. of eadxm m C~<>-£*<> bonding environment was higher to Surfaces 2-4, Surface .1¾ and Primaria!M surface than in the other surfaces: analyzed, The proportion of carbon in GC);? -bonding environment was higher in Surfitees: 2-4/Burfaee 13, and CellBIMD1^ surface fb;an,iB the other surfaces analyzed:, The proportion of carbon in, OO bonding environment washigher in Surfaces 1 -4, Surface 13, and CellBtNDsurface than in: the other surfaces analy zed. The proportion of carbon in CrfOj-C bonding environment was higher in Surfaces 1-4, Surface l3>:::CeilB1ND!M surface, and Primaria3 Surface than in the other surfaces analyzed. The energy loss peak resulted from an aromatic Π····> if transition, and is an indicator of surface aromaticity, ff 2:2?| The 01 s spectra peaks were almost Gaussian: and could not be curve fit, Ml s spectra peaks were curve fit (best chiasguared fit), ip, order to identify andtpamify the bonding environments for nitrogen in the surfaces, by using peak widths and energy locations for species as found In the Sitemturo fTable 9), 'Phe eOneentrafions are t^fdrf^iTteaiBikof atomic percent, which w"ere.c»btamedby-:m«i%i^tg'^e:area. percent by the atomic concentration. The Mis signals fYorn Nunc ion I>idialM, GellBIMB^, GostariM, and Palcon{M surfaces were weak, aud it was, therefore, not ; possible m do idestificatfon of the bcutding environments for nitmgen in these surfaces. M is spectra were indistinguishahle for surfoee rnodified plates 1-4 and 13, and dataresulting from curve fitting of two representative Mis spectra is shown . The proportion of nitrogen in -NHrf bonding environment was higher in Surfaces 1-4 and 13 than in Surfaces 14 and 15 and Primaria/^ surface, hfitrogoo fo'“K&amp;:-bonding environment: was detected only in Surfaces 14 and 15 and thimsriaiM surface. Nitrogen ih .-140¾ bonding eoyirondteni was detected, only in Surfaces l~4 and 13, and in .tpslagferMmpfoof Surface .14 Mitogen in-•MOr:bonding:envixwnment was ddfoctediOnly in Surface 15 add; Primaria‘M Surface,; f022fl| CellBlND1^ has previously been described as having an elemental composition of 70.4% carbon, 29.0% oxygen, 0.b% nitrogen, and <0:01% other elements, and a relatively high. coscentraHonef €«[0|£, CM3, and CO0H/R as analyzed by ESC A [Cfomisg Technical Report (2005), Ctfoving# CeiiBIND# Surface: Ah Improved Surface for Enhanced Cell Attachment (CES-AN-05? REV I) on hdp://eataIog2,coming.com/Lifoseicncesrtnedia/pdi71 Cel IBIND; Impmved. Surlkec; CLSwAN057 .pdf], |0229] Prituarit—'has preciously been described as having m elemental composition of 74.6¾ carbon, 14.1¾ oxygen, 11.1% nitrogen, and 0.2% other elements, with mainly nitrile (ϋ=;;Νΐ and urea [ΗΜ(ί><>)ΝΜ] earbon-tmmlrogen 44 analyzed b) ESC A.

Aimtiip Fan c 4 licroscopy (AFM) [0230] Surface modified plates 1 -4 and 1.1,and plates with. N unefon DeltaiM and CellBINDSM saffaces were analyzed using AFM., Samples wem ahalyKcd using a Digital Instruments Multimode Atomic Force Microscope in tapping mode. The tip used was a tapping mole tip, type 1 ESP7. Samples were attached to the sample disks with double sticky tape Regions: of Id pm x 10 pm and 500 nm x 500 nm of the sorfoee--treated part of the plates were analyzed. Surface mean roughness (Ra) and maximum iKiuhtfRmaMfo units of nanometers are shown:: in Table 10., Like the plates with. Ntmcion Deiia:iM and CeilBIND -surfoceSj surface modified plates 1-4 and; 13: wem folatively smooth, and Ra and. Rmax did not correlate with surface treatment: time in either of the: t;wo: «cans. Analysis of non-treated polystyrene and: oxidized polystyrene surfaces intended for cell culture, and PnmarialM surface ltas been described by Slieo and Hptheit (j. Biomed. Mater. Res. 57:336-345,2001): surface roughness approximately 4 nm for all three surfaces.

Example IS

Surface Elemental Composition and Contact Angle in Eelatteo fu llutua» ES Cell Attach meat and Colony 'Formation.

(0231] A summary of the results of the: 3CPS analysis of surface ekmeutal composition, the surface contact angie measurements, and fnnnan ES ceil attachment and colony formation experiments is given in Table 1L 18232) Human ES cell atlacEmsut ίο a»d colony forrnationCat least 15 colonies per 10 cm" surface) on a solid substrate surface to the absence of a compound capable of inhibiting Rho or Rk) li«e«« observed on only sttHace nfodified plates 2-4 apd •t'l, CellBlND1M plates, and PnmariaiH plates (cells were presented to the surfaces as a suspension of clusters OFeelis). Surface modified plates 2~4 and 13 supported cell attachment, colony formation and passaging. After about three passages, the growth rate of hntnan ES cells on surface modified plates 2-4 and 13 declined spontaneously (only in the absence of Rbo inhibition and Rho kinase inhibition), although cel! morphology indicated that the cells were not difiemntiatrng. Furthermore, piuri potency marker expression was maintained in cells passaged lour times bn Surface 3, CeIIBHMI>iM plates supported human ES cell attachment and colony formation, but differentiation of the cells wus observed prior to the first passage.

Based upon cell morphology observations, ibimafiaiM plates supported hnthan ES cell attachment and colony formation, without signs of differentiation (passaging was not tested). Both oxygen (for example. Surface 2 versus Surface 14) and nitrogen (for example, Primariam versus Costar:M; and Surfaces:! and 13 versus CellBlblDiM} content of surfaces had an effect on the ability of the surfaces to support human ES ceil attachment and colony ibrmaiion lit the absence of Rho inhibition and Rho kinase inhibition. Surfaces with a nitrogen content of at least about 0,9%, a sum of nitrogen; and oxygen content of at least about 223%, and a water contact angle of at least about 13,9 degrees supported human ES ceil attachment and colony formation in the absence of Rho inhibition or Rho kinase inhibition. (02¾¾ Human ES cell attachment and colony formation (at least 15 eblomes per 10 cm" Surface) on a solid substrate surface in file presence of a compound capable of inhibiting Rbo or Rho kinase was observed oh surface modified ,plates 1-15, surface modified, plate 19, surface modified p!ate33, surface modified plate 34:, CtellBlMD1^, aftd Frimaria1^ (cells were presented to the surfaces as a suspension of oldsters of bolls). We: noted that, surfaces 2-4 and 1 S and Prinisri.aiM were better:than surfaces 1, 1.9, 33 and 34 and CeliBINPm, which again wemfoetter than.surfaces ,5-12,14 and 15, at promoting human ES cell attachment and colony formation. On surface prodified plates 3 and 4, and in the presence of a Rbo kinase inhibi tor, human. E$: pells attached and formed colonies that expanded and could be passaged at least Iff : times, giving rise to pluripotent cells with normal karyotype (karyotype tested, ©foy in .cells grown cm Stirfleo 4), Βοώ oxygon (for

Deifor^ and nitrogen (for example, Pdmaria5 M versos Costar^; aocl Snrface$ 2 and 13 Versus of surfaces had an effect on the ability of the surfaces to support human ES cell attachment and colony formation in the presence of Mho kipase inhibition, Surfaces with a nitrogen content of at least about §.53%: &amp; simipf nitrogen andtoxygen content of at least about 17.2%, and a water contact angle of at least about 13.9 degrees supported human HS cell attachment and colony fonnation in the presence of ;Mbo kinaseuBhlBitioti Surfaces with a nitrogen content of at least, about 0.5%* a sum. of nitrogen and oxygen content of at least about .1713% hut less than 19.9%, and a water contact bugle of at least about 9.4 degrees supported human, ES ceil attachment andeofony formation In the presence:of Mho kinase inhibition in some cases (surface 14% hut not in others (surfaces 22-24|. 1112341 We noted that remo vat of Mho kinase inhibitm fkuu human ES: cell euhures cultured on ..surface modified plated resulted in defoehmomof the human IS cells from the Surface of the solid substrate. The ceils: could then be reattached to the surface by re-treatment with a Rho kinase inhibitor. Cilvon thai enzymatic pasMge of human ES cells is a potential stressor and may cause karyotypic instability, using temporary mmovdi of Eho kinase InhiMfor to passage human IS ceils could eihrhnato the stresses: of enzymatic passage, |023il Human:ES cell attachment:and colony formation was ai so demonstrated using ammal-emnponenhfree'medium*. Mho kinaseifoblhiiien andxurtkee modified plate 4. Ihe-: treatment of surface modified plate 4 with eMraeUllUlar matrix proteins resulted id more colonies., but only in the presence of Mho kinase inhibition, {112361 In addi tion to passaging human ES cells with enzymatic methods that maintain colony style culture conditionsby passaging cells as dusters, human BB: cells: could also be passaged as single cells using enzymes like f rypLE™ or Aecufose1**, In foe presence or In the absence of Rho kinase iohibitoiy human ES eel! colonies dissoellted. info a suspension of single cells using TrypLEtM attached to surface modified plates 3 and 4, and formed eolonies that could be passaged at least five times and give rise to cells with pluripotency markers. 10237) Removal of Rho kinase inhibitor from the human ES ceil cultures prepared by passagmgthe eelis as a suspension of single ceils did not result in detachment of the hutpan;:ES:celi^fcmdhe.Suffaee of the solid substrate, hot resulted in colonics that grew foster than if the Rho kinase inhibitor was not mmoved.

Example 19

Treatment with Y427632 Enhance 11EK293 Cell Attachment to Surface Modified

Plates |0238j Human euforyonie kidney ceils 293 (HEK293,ECACC no, 85120602) were maitttamed ih Eagle's Minimum Essential Medium (EMEM; Lonaa, Yefeiers, Belgium) eonfoinlng 10% fetal bovine serum (FBS; Lonza). The cells were adapted to Pro293a.~CDM medium (Lonaa), a chemically defined, serum-free medium optimized for enhivaflon of adherent HEK293,, hy gradually and over several passages using the sequential ratios of 3:1, RE 1:3,1:7, and finally 0T of serum-supplemented EMEM and Pro293a-€DM medium. For maintenance and adaptation, HE K. 2 93 cells were seeded at 2,0 x 1 (1* cells/coY in 73~cmr flasks with Nunelon Delta1 M surface (Thermo Fisher Scientific, Rosfeilde, Denmark) and passaged at 70^80% confidence using Trypsin/EDTA for dissociation, 10239] Ero20teC'DM medium. (100 id) supplemented: with Y-27632 (Sigma Chemical Co., St, Louts, MO) in. concentrations of 1,0,4.0: or 10 p.M was dispensed; in flat-bottomed;,: 96-weil plates with Surface 4, Nuueion Delta1** surface, or CeliBIMDiM surface. Another 100 pi of Pro293a-CDM medium with ΉΕΚ293 cells was added io the;wells: (4,0 x .104 eeil?denr), The: cultures were then incubated at 3.7*0dp a humidified atmosphere of 5% €(¾ in. air for: (i) 96 hours; or (if) 48 hours, followed by washing cultures once with 200 μ! DtilheecoY Phosphate !3«ficrcd Saline (DPBS: Lonza), then: adding 200 μΐ of Fro293;a~CDM medium with:cmi:Y-27632, and finally incubating cultures for another 48 hours, j.0240] The number of viable cells in the wells was then determined using a lactate dehydrogenase (LDH) activi^' Mt fiom Eoehe, S witeerland; Briefly* wells were washed with; Fto293a~CDM medium, and adherent cells Were lysed in 100 μ! DPBS with 2% (v/v) Triton X-100 (Sigma Chemical Co.) during a 30-mia incubation at 3?°C> Xysafe and lOO-pl catalyst and dve reagentmixture were mixed and incubated id the dark at 25-CHbr 3© nun. The reaction was stopped by adding 50 pi of i .0 M BXL and the absqfoance at #0 nm wai measured in a Ifro;

Teea% Austria). The number of ceils was calculated using the A490 values from these samples and from standards containing LPM from a knomt number of cei ls. |824lj The effect of the solid substmte surfaces and ¥-27633 on attachment and growth of BE£i93 ceils in Pio293a-€BMMedium is shown la figure 31a, where the 96-hour continuous exposure to Y-27632 is labeled44 Y-27632 96b oif5 and the 4BT«>ur eoatinuous exposure to Y -27632 followed by a change of medium and 48 fronts of ineiibation In the absence of ¥-27632 is labeled “¥-27632 48h onftfbh off*, la the absence ©FY-27632. ifEK293 cells attached to all three surfaces. A change of medium after 48 hours of incubation resulted in significantly fewer cells in the cul tureSv ineasured sfrer 96 hours of incubation. Y-27632 enhanced attachment of BEK.293 cells on Surface 4 and CellBil9D1M surface when applied at concentrations of 2,0 and 5,0 μ.Μ Rethdving Y-27632 after 48 hours of inenbatton resulted in signiEcant detachment of eel Is from all three surfaces...

[0242] A similar experiment, but using 2,0 x 1:04 non-adapted BER293 cells per cnf and EMEM supplemented with 10% EBSdhroughout, was performed. The effect of the solid substrate surfaces and Y-27632 on attachment and growth of 11EK:293 cells in EMEM supplemented with 10% PBS is shown in Figured lb, where the 96-hour eotftinuons exposure to Y-27632 is labeled “Y-27632 96h on” and the 48-hour continuous exposure to ¥-27032 followed by a change of medium and 48 hours of incubation in. the absence of Y-27632 is labeled 7 Y-27632 48h onMSb ofP. In the absence of Y-27632, HEK293:cells: attached to all three surfaces. A Change of medium, after 48 hours of incubation resulted in. significantly fewer cells in. the cultures, measured after 96 hours of incubation, Y-27632 enhanced; attachment of HEIC293 cells on Surface 4 and CeIIBIMDm surface when applied at concentrations of :2,0 and 5,0 μΜ, Removing Y-27632 after48 Imim of incubation resul ted in significant detachment of cells front Surface 4 and CellBlMi>m

Example 2d

Treatment with Y-27P2 and B-I152 Enhance ΠΕΚ293 Cell Growth 0» Surface

Modified Plates !©243j HEK293 ceils wem: maintained i»: EMEM|Lorsza) containing 10% F BS (Eon?®),·.

Ceils were passaged at 70-80% confluence using l^psin^BD'fA.lof dissdeiaiion, and seeded at e 2,0 x }Φ cells/enri in 75-·οηΥ DejtaiM surface (Thermo fisher Scientific, Rosldide, Denrnart|, |0244| EM EM (500 pi) supplemented with 10 % PBS containing 1.0, 5.0, .10,15 or 20 jtM Y-27632 (Sigma ChemieaIi:o,ior 0.4,1-2,:1.6,2.4 or 2.8 uM H- l 152 (Calbiochem, EMB Chemicals Inc,, Darmstadt, Germany) was dispensed in MiiMdish 24-well plates with cither Surface 4 or a non-treated (but gamma irradiated; 25 kCy ) polystyrene: surface, Another 509 μ! of EM EM supplemented with 10% FBS and containing EEK293 cells were added to the wells (2.0 x I O'* cei!s/crn\ The cultures were placed in. an IncuCyte—1 Plns:(Esse« Instmments, Michigan, USA) and Incubated at 37°C in a humidifiedatmosphere 0.25% €(½ in ah. The lneuGyteiS,i Plus Is an aufontaied/misging platferpv cotiflgureddo lit inside a ( 7.½ incubator, and designed to provide;:kinetic,; non-lnvasive:live ceil imaging by acquiring phase contrast images of the cells at user-defined times and thehuliunesl.

The primary metric of the in strumeat is cultureconfluence,, that is, the fraetiott of the surface that is covered by cells. The BEK293 cells were incubated for 72 hours without manipulations, and images were collected every two hours at 9 positions in triplicate cultures. Culture confluence was determined using the IncnCyte3M Plus software (v. 3.4 J, 25966). |0245] Increasing concentration of ¥-37632 and H-l 152 enhances attachment and growth of BEIQ93 cells on Surface 4 (Elgnre 32a). The effect of a nom-treated cell culture surface and Y-27632 or H-l 152 on attachment and growth of BEK293 is shown in (Figure 32b). Growth and ahaehrnenr of HEKi293 cells was slightly enhanced in the: presenee bf 10 plrf Y-27632 and 0.6 -1,2 uM 14-11:32. However, the ehhancemeni of growth, and attaehmenf of H:EfC293 cells on a oon-trcatedeell culture surface is insignifieant in. comparison to 'Surface :4.

Example 21

Tptatmesii with 11-1152 Enhances IIEK293 Cell Growth and Attachment ίο

Surface Modified Plntes t$246| BE1&amp;293 cells were maintained in EMEMfLomsa) containing 10% F BS (Eouza),

Cells were passaged at 70-80% confluence using Tr^in/ED'fcA. for dissociation^ and seeded at c 2.0 x; 104;eeils/efif: /in IS-enfi flasks with Mimcion Delta^^^surfece (Thermo Fisher leiemific5 :Roskildes I>ournark}. |:0247! EMBM (1.0 ml) supplemented with. Ml % BBS coatakmg 0.4, 0,8, 1,,2,. 1,6,2,0,2,4 or 2.8 μ .VI H~ 11:32 was dispensed in Midtidish 12-well plates with Surface 4. Another l.Offilof BhfEM supplemented with: 10% FBSand containing ΉΕΚ293 celisWere added to the Wells (4.0 x KP ceOs/cun. The cultures were piaeedinnh lneuCyie!M PiaSj and incubated ai fl? C in ahiimidifM.ntk©sphef8:©l‘5% €€&amp; itt&amp;ir S*f 42,hours: (images were collected' every 6 hours}. One ml of culture medium was then removed by pipetting, and 1,0 pi EMBM supplemented/with 1.0·% PBS containing 0.2,0.4,0.6, &amp;&amp;* T .0, 1.2 and 1.4 μΜ 1:1-1152: was added'. The cultures: Were placed in the itteu£:>1eiM Pius again, and images Were collected every hour over the following 25 hours. Images were c«lJseted .at: culture confluence Was determined using the :lneuCpe1M Pius software. Mages from the lneuGyiem Plus eolleoted at speeiie positions in MBE203 cell cultures grown m the absence or presence OTB-T1S2 (0Μ μΜ) was retrieved and presented as phase-contrast micrographs tor the comparison of HEK293 culture morphology at the Mfldwing time points: start of incubaiion (0 hours), just before medium change (42 hours), 1 boisrsftertfee medium change (43 touts), and, finally, after 52 bouts of incubaiion. I1I24S] In the absence of B-l 152 and m the presence of :0,2: μΜ or Q.4 μΜ B~ I 1:52, the change of 50% of the medium after 42 hours of incubation resulted in a significant mduction in cultmw confluence (Figure 33a). In the presence of 0.6 μΜ, 0,8 μΜ or 1,4 μΜ H-l. 152. the effect of changing the medium was nunirnaL BES093 ceils grown on. Surface 4 .lathe presence of H-l: 15.2 cohered the solid substrate surfaces more evenly than BE&amp;293 cells grown on Sutfaee 4 in the absence of B-1:152 (Figure 33b). in the absence of Η-1152, HBK293 latijgel^iujst-er^, whereas, IIBK.293 cells in; the presence of B-I 152 formed smaller el asters with lower cell density.

Example 22

TrsatMeM wMh Y--27632 Enhances ilEOftS Cell (irowth TBree Passages «a Surface Mudilfod Ftetes |0249] EMEM §m pi) supplemented with ! 0 % PBS eoffidintag 5.0 μΜ Y-27632 was dispen^dm wcllfbf Mdlfidish 24~well plates with -Surface 4 or Nunclon De!taiM surface, Another 500 μί of EMEM supplemented with 10% FBS and eothalning BBK293 ceils was added to the weds (2.0 x ;Mf ceils/emfi, and the endures were ineuhated at 37¾ in a .humidified atmpsphem'of $%:£02.m air for 3 days. Ceils were passaged by treatment with Trypsm/EDTA (Bourne Verviers, Belgium) for two minutes at 37-0, and the total cell number was determined using a KpclsdCoutb Ceil Counter (Chemometec A/$, Altered, Denmark). For successive passages, BBI093 cells Were seeded at 2.0 ¾ 10s* ceils/emf The growth of M1R293 ceils On Surfeee 4 and Himelon DeitaiM sortaee was enhanced by the presence of 2,5 uM ¥"27632 (Figttreddf

Example 23

Attachment, Cultivatkm and Maintenance oMlmmrn Embryonic Stem Cells Using Bur foee Modified Plates 4, IS, and If that leek Extracellular Matrix f roteta/Componeois and Feeder Cells 102501 Passage 42 B1 iiES cells maintained on i :30 MATRICES coated plasiicwafo: in MEF conditioned media supplemented with 8ng/ml. of feEGF were lifted by LS BERASE™ eaxymatic treatment and plated to surface modified 96 well format plates at a 1 to 2 dilution in MEF conditioned modia mppiemcoted with fng/ml ofhFGF. The cells wereptaied to modified surfaces 4 f ifoor 19, or Frimariaw. In order to determine the effect of Rho Kinase inhibition on bindihg to the modified surface we treated the cells with either ΐΟμΜ of the fobo Ki nase inhibitor ¥-27532, or 3 or ΙΟμΜ of the Rho Kinase inhibitor Efl 152glycyl. Untreated cells served as controls. Afler 24 hours In culture the wells were aspirated, the cells were dried, and the wells were stained with Crystal violet 10251] We observed that after 24 hours in culture, ES ceil coiontes had attached and spread when treated with Rho Kinase inhibitor!: oh surface mpdtFed plates 4 and 19 and the

Frmiari#** plafe, however the same efieetwas: not observed on surface modified plate 18 (Figure 35).

Example 24

Attachment CHltsvutkm »&amp;ά M&amp;Mfemm^oiMumAn Bmbt^mk Slxm €ellx l lsisag Sorted Modified Plates 311,31,32,33, and 34 that Sack Extracellidar Matrix Prpt^ia/CdaipoMats and Feeder Cells

1.0252] Passage 47 HI hES cel lx maintained on 1:3(1 MATRK3EL coated pksticware ia MBP conditioned media supplemen led with'Bog/rni of bFGF were lifted by Ι^ρΕΕ®** enxymatk treatment and. plated to surface modified 96 well format plates at a 1 to 3 dilution:®: MBf conditioned media supplemented with Etig/ml of bFGF, The ceils: were plated to toodified: surfaces 30* 3K 32, 33, or 34. In order to determine tbe^iect oi,Satd.Mpasd'mhi|iitkm on binding to fte modified surface we treated the ceils with 3uM of the Rho Kinase irkibdorTI-1152glyey:i, Untreated cells served as controls. Additionally, ceils were seeded to wells in the surface modified plate that were pretreated with Mstrigei ™ 24 hours afe plating the media was changed with fresh MBF^pdtiopdd' medlkapppl^ihented Adih'%g$al nfihfAiF,ond for cells seeded in the presence of the Aho Kinase inhibitor the media was supplemented wi th 3μΜ H-1 ISSglyeyt Aftef 48 bonfsiM culture the ;wclis:#erhaspifated5 the cells were dried, and the wells stained with Crystal violet. I d253| We observed that a tier 48 hdum in culture, ES cell colonies had attached add. spread when Rested with Rho Kinase: inhibitors on surfime modified:: plates. 33 and,34 (Figure: 39 and 40 respectively), however the same effect was not;observedon surface modified plates 30,31; or 3i (Figures 36-40 respectively^ ARachmeoE Cultivation anti Maintenance of llama» Embryonic Stem Cells

Using Sorloce Moilliccl Plates 22,, 2&amp; 24 or 29 that lack Extracellular Mates Proteia/ComponenH anti Feeder Cells 1(1254] Passage 46 HI hES cells maintained on 1:30 MATR1GEL coated plasticwaro m MEF conditioned media supplemented with Bng/nll of bFGF were lifted b> Liberase™ enzymatic treatment and plated,fp.: surfacemodified bDmrn dishes at a: 1 to 3 dilution id MEF conditioned media siipplentented.with 8h;g/ml of bFOFl The ceils were plated to suAed idodified plates 3,4, :2¾ 23,24 add 29, In order ip determine the effect of Eho: Einaseiuhibition on btfidifigto the modified surface we treated tfee cells with 3u:M of the Ilho Kinase inhibitor p~ I I52glyeyl to plate the cells. The media was; changed with fresh. MEF conditioned media supplemented with: hugfitfi of bFGF and: 1 μΜ of the Rho Kinase inhibitor |3~11.52giyeyl 24 hours after plating the cells. Cells seeded to modified surface 3,4 or matrigel/^ coated plastic served as controls. The plates were observed by phase ipicroseopy 24 and 4B hours after plating. We observed that:after 48 hours, in culture, ES cell colonies had not attached to surface: modified plates 22,23,24 or 29 plated with or wdthodt Rho Kinase iuhibitop while cells plated, to surface modified plate 3 or 4 in the presence of Rho Kinase inhibitor did attach and spread.

Example 26 itif titer Surface Characterlaation of ihe Surface Modified Pftdes of tlie Wmint

Invention IVatcr Contact Angles |12551 Surface modified plates 1-4 and 13 weto individually packed in plastic hap, stetflked, and stored at room temperature througbont a 4i}-week test period, Contact angles were first measured one week after surface treatatent and sterilkatfen, and then again ml dm time points given in Figure 41, All contact angle mcasnrements were done as described in Example 17, Measurements on NunelOn DeitarMaod CeilB!KDiM surfeees was perfbnned under the same experimental conditions as measurementa on Surfece 1-4 and 13, bnt the surface treatment and sterlliaation was done more than 12 weeks before the fimt measnrement (Nuelon Delta was sterilized one week before the first mcasttrement). Figure 41 shows that surface modified plates 1-4 and 13 were of similar h.ydrophiliciiy and more hydrophilic (lower water contact angles) than Bunclon Delta5 M and Ce!IBiNDm surfaces, 1¾ hydrophtiietiy of surface modified plates 1-4 and 13 waa stable ibrat least 41 weeks after smdaee treatment and sferi Itzafion, 10256:) Clontaet angles were: also measured on surface modified plates 5-1.2,22-24,29,30 and 33, which werepaelced in plastic bags,: sterilized as described in Example Id, and scored at 9 weeks (except for surface modified plate 29 which was stored:for 28 weeks). Snr&amp;ce modified plates 18, 19, 32 and 34 were in single-microweli fomrat and could, therefore, not be used for measurements of contact angles. Surface modified plates 30 and 33 were in a micro well plate format, and contact angle measurements were perforated on the backside of the plate and not inside wells. Contact angles were measured as described in Example 1? (for the highly hydmpfnlic surface modified plate 29, a smaller drop of 2.5 μϊ MilllQ water was applied), but triplicate samples were analysed, with 7 drops being applied per sample. Measurements on plates with Costar5 M5 Faieon:iM, PrimariaiM andfNunelpn Delta— surfoees was: performed under the same:experimental conditions, but the surface treatment: and sterilization: was done more Ilian 12 weeks before the first measurement. Figure 42 shows that surface modified plates 5-12 were more hydrophilic (lower water contact angles) than Nuncfori; 0e3fo^

Falcon ^Surfaces, The hydmphilioify of surfaces 542 was comparable to the hydrophilicity of the PrimarialM surface, and higher than the hydrophilicity of surfaces 'fi4 and 13 (shown in Figure 41)< The hydrophilieity of surface modified plates 22-24 and 33 was eompamble to the hydrophilicity of suriices 1-4 and 13: (shown hi ligure 41), Whereas the hydrophiJicity of suffdc&amp; Sf) was comparable to the hydfophilicity ofNnnelon Deltam, Cos(arlM and ibikx)nTM surfaces. Surface modified plate 29 Wiis significaotly mom hydrophilic than the other surfaces analysed.

Negative Charge Density | 02S?1 The densi ty of negative charges on surface modified plates 3-12 (all 5-em dish format), 18,19,3% 32, 33 and 34 (all Merowell format), Mf^ri|-h0cidified ;plai#-22-24 and 29 (all 6-cm dish format), and Γο11Β1ΝΟίΜ surface (3-cm dish format), Prifnaria^ ,mrf#e;.(multidi8hr^:'^rfn0f -00¾ KuttcJon. Delia™ surface (3-cm dish format) was detennined. Agueous erystal violet solution (0.015% w/v) in excess was add ed fo each format (0,34 m !/cm2 for di sh format and 0,13 mi/cur for mierowell format), and was incubated for 60 minutes at tooth tebfoerature under gentle shaking (50 rpm). in order fo remove crystal violet not bound to the surfaces, the dishes: were washedthree times , with 3 ml MilIiQ wafer for the dish formats and three times with 350 μ! MiiliQ water for raierowcli formats, and then, dryed over night at 60°€. The Crystal violet bound: to the mlrfce was desorbed by addition of 0,1b ntl/ehr of 0,1 M Bp in EtOH solution (99%) and ihepMfing the dishes for 2 minutes at Mom temperature under gentle shaking (50 rpm). Absofounce of tee HChEtOH solution with desorbedbrystai violet was Measured at iping)ah,:inVisiofo2.1 Q0 nieropiate reader (Perkin Elmer; Waltham, MA, USA), Absorbance values were cormctddifof^ek^nnd^hsotrbanceof B€i :EtOH solution. The negati ve charge: density ^•^eashj^.OMlhiee dfeh.es with shrfece modified plates 5-12,22-24,29, CeilBOn0|Ms Primaria™ and Munclon Delta™, and absorbance Measurements were performed in triplicate for Mach dish. For fa]f^ce.inh^bdpid^l;^» 19, 30* 32, 33 mb· 34, one: sample was tested: with triplicate measurements, [0258] Hnnegative Charge denshieStef surface modified plates 5~1.2wttee similar., and those surfaces had significantly low# negative ch.#ge:: densities Man CellBMD™ arrface and Nunclon Delia™ surface*· but significantly higher negative charge densities than the Pfknaria™ surface (Figure 43). The negative charge densities Of surfoee modified plates i§i 33 and 34 were significantly higher than the negative charge demities of surface .modified plates iff 30 and 32, the latter heteg the respective nonAreated surfaces in Me same pOlynter material., The negative charge densities of surface modified plates 22-24 and 20 were normalized to the negative charge density of the Nuncfon Delta1*1 surface. and Figure 44 shows that surface modified plates 22-24 had higher negative charge densities Man ihe Mundon. 'Delia’ M surface, whereas the negative: charge density for surface modified plate 29 was sigruficahtly lower than Me negative charge dcn>ay of MeNonciOn Delta1·'*' surface (and surface 4). A-fo?y PMotoeieetnin Spe&amp;twscopy (W$) I0259J Surface modified plates 5-12, IB, 19,22-24, 29, 30, 31-34 were analyzed using XPS as described in Example 17. Surfoce elemental composition in un its of atomic percent is shown in Table 12. All surfaces coMained carbon, oxygen and nitrogen (hydrogen is not detected in XPS), except siirteco n»diiied plates 31 atid 32 (not plasrna treated), which did not contain nitrogen. Surface modified plates 5-12 contained less oxygen than sur&amp;ce modified plates M- and 13, but s%nlfieantly rnore oxygen than Dostar™ Falcon™ and ;]%nclon Delta™ surfaces (shown. M Table 7), Surface modified plates 5-12, were prepared by microwave plasma treatment ivhiieiiuffaee modified plates I- 4 and B wempmduesd by eorena plasma treatment Siirlacg modified plates,.19, 33 and 34, which were prepared by Cdietta:fii^ii;ifreaimen^ but injection molded from other polymers: than polystyrene (which was dsed in the preparation of surface modified plates 1-4 and 13)* contained oxygen levels comparable to those of surface modified plates 1-4 and 13.. Surface tnodified plates 22-24 contained less oxygen than surface modi&amp;d plates I “4 and 13; Surface modified plate 29 contained oxygen at a level comparable to aurikce modified plates I -4 and 13. Surface modified plates 542, 19,33 and 34 contained less nitrogen than surface modified plates 1-4 and 13, hot more ni&amp;&amp;gon FalconiM andb&amp;nclon Delta — surfaces (shown in

Table 1% Surface modified plate &amp;un die other surfaces analysed, including the Pri.mana!M surface. |026O] Os spectra peaks were curve fit (best chi-squared fit), in order to identity and quantify the bonding environments forcarfmn in the plates, byissihi peak widths and energy locations for species as found in the literature (Table 13).

The concen tratiohs are reported In units of atomic percent, which were obtained by multiplying the area percent by the atotnic concentration. All plasma-treated surfaces, except surfaces 10, 22-24 and 29, were similar in terms of the carbon bonding environments. The proportion of carbon in €4©J~C was sigpificaotly higher in Surfaces 19, 33 and 34 than in surfaces 5-12. IK, 30 and 32 and surfaces I -4 and 13 (shown in Table 8). The proportion of carbon in O- j’C:::OJ -O bonding environment was lower in surfaces J-12, 19, 33 and 34 than in surfaces 1-4 and 13. The proportion of carbon in was significantly higher in surfaces 5-9, 11,12, 19,33 and 34 than in surfaces 1-4 and 13, but comparable to^Nunclop Dcham and GellBIND*^ surfaces. The proportion of carbon in fi-O-Cof C-44¾v bonding environment (same energy locaiionin sgeetnfiwas lower hr surfaces 5-12, 19,33 and 34 than in surfaces 1*4' and 13, hut was higher than in Corfat^, Falcon **\ CellBfND'^ and PrimanaiM surfaces. The proportion of carbon in C-O-G*-© bonding environment was higher in surfaces 19, 33 and 34 than in surfaces 5-12, and comparable to the level in surfaces 1-4 and 13. The proportion of carbon in C:::<) bonding cnyitonment was higher in surfaces 5· 12 than in surfaces 19, 33 and 34, but lower than in surfaces 1-4 and 13, The proportion of carbon in CDfi bonding environment was higher in surfaces 5-12 than in surfaces 19, 33 and 34, and comparable to the level in surfaces 1-4 and 13 , Surfaces 22324 were similar iu terms of eaitea bonding eavimmects. The proportion of carbon in C---{0}--C. 0 |C' ::0)···(), G~0--G orC-NBy ^€-0--0=^0 andfM} for surfaces 22-24 was significant lower than for surfaces: 1-4 and 13. Thepropottionof carbon in €1(5?' and C*~C~&amp;-C-Cm bonding environments was higher for surfaces 22-24 than for surfaces .1-4 and 13'.

The carbon bonding eovlfonment of sutfece 29 ms different from the carbon bonding environment of all other plasma-treated surfaces. The proportion of carbon in C-{0}~ G was comparable to surfaces 1-4 and 13. The proportions of eaiftonin Q- (C;:::Oj-D, ( Of and - bonding environments were lower for surface 29 than for surfaces t~4 and 13. The proportions of carbon in €™0~C or C--NHI'',; 0-Ό~0Φ“0 and CTO bonding environments were higher for Surface 29 than for surfaces 1-4 and 13. The energy Loss peak remitted 'from an aromatic fl-oll* transition, and is an indicator of surface aromaticity.

[02¾¾) The 01 sspeeim peaks were almost Gaussian and could not be curve fit.: N1 sispeetra peaks were curve fit (best chi-squared fit), in. order to identify and quantify the bonding environments for nitrogen in the surfaces, by using peak widths and energy locations for species as found in foe literature (Table: 14), The concentrations are reported in units of atomic percent, which were obtained by multiplying foe aim percent by foe afomie concen tration. The proportion of nitrogen in~49BT bonding environment in all surfaces, except surface 9, was lower than in surfaces 3M and 13. Nitrogen in bonding en vironment in surfaces 5-12,19,33 and 34 varied, but was higher than in surfaces 1-4 and 13, Nitroge»tft'”^02.1x)»di»g.'dnvir0nfoent«fi surfaces 5-12,19, 33 and 34 variM,lbut was lower foan tn MrfacesT-4 and 13. Nitrogen iu -NOs bonding environment in surfaces 5-12. 19,3¾ and 34 varied, but Was higher than in surfaces 1-4 and 13, The nitrogen bonding;environments of surfaces 22-24 and 29 were different from foe other plasma^treated surfaces. The proportion of nitrogen in 22-24 and 29 varied, but was significamfy higher than in surfoces 1-4 and 13, The proportion of nitrogen in --N().> bonding environment was lower in surfaces 22-24 and 29 than in surfaces 1-4 and 13, The propottion of nitrogen in 05::G--N-G::s:0 bonding envimmuent was comparable in surfaces 22-24 and 29 and surfaces 1-4 and 13.

[0262] Publications cited throughput this document are hereby incorporated by reference In their entirety, Tthhougb thevarious aspects of foe invehtion have been illustrated above by reference to examples and preferred embodiments, it will be appreciated that the scope of the invention is defined not by the foregoing description but by the following claims properly construed -under principl es of patent law.

TABLES

Fable 1 Expression of Piuripotency Markers in Ceils of the Human Embryonic Stem Cell Line FI 1 at Passage 50., Cultured on the Surface Modi lied Plates of the Present Invention

Table 2: Expression of Pluripotcncy Markers in ('ells of the Human Embryonic Stem Cell Line Hi at Passage 53 Cultured on the Surface Modi tied Plates of the Present Indention

Table 3: of Msfers Characienirtfc of the Definitive Endodenn Lineage in

C'ell| of the ffunian Bthfetyomc Stem Leli Line HI at Ihispge 51 (p2) and Passage 53 (p4) Cultured on the Surface Modified Hates of the Pfcsent Immotiom Tmated with Activin A % Expression of the surface marker CXCR4 following differentiation of 11 i human ES ceils to definitive endoderm

Table 4: Percent eonflnenee (acquisition area occupied by objects) and iota! human 119 embryonic stem ceil colonies greater than 50K sq. microns in the acquisition atm after one passage on the Surface Modified Plates of the Present Invention

μΜ concentration of Y-27632 for 96 hoar Ri exposure: culture

Table 5: Exp region of Markers Charaeteristk of the Defimffve Bndoderm Lineage in Cells of the Muni'a»:. Embryonic Stem Cell line Hi aCPassage 51 (hdtured on. the Surface McdMed Plates of the Present invenite. Treated with Activity A % Expression of the surface marker €X€R4 following .-differentiaiioft of 111 kutnen ES ceils to deEnitive endoderm

Table 6: Preparation· of the Surface Modified Plates of the Present Invention

* Not sterilized by gamma irradiation

Table 7: Surface Elemental Composition of Surface Modified Plates as Determined by X PS

* Other elements were detected at a concentration of 0.4%

Table %: Carbon Bonding Eaviroameat by Cis Spectra. Carve Fitting

* The functional group was identified in only one sample.

Table 9: Nitrogen Boa drag Environment by Nls Spectra Curve Fitting

* NTs spectra were irsdishayuiidutblo ibr Surface modified plates M and 13, and data resulting irons curve tit of two representative Nls spectra is given, ** The functional group was identified in only one sample.

Table 1(1; Surface Roughness of the Surface Modified Plates of the Present invention, as Determined by A.FM

Table .11: Summary of the Results of the XPS Analysis of Surface Elemental Composition and Homan Embryonic Stem Cell Attachment and Colony Formation Experiments on the Surface Modified Plates of the Present In vention

idmatSiXt t>f fm than i 5 cd&amp;xK** p<*r Hi m’4 r,*\ Λτ-> Λ, a&amp;d v-t--t--t-vr autra <15 «ιτ«κί·.« per 50 em'k myrty aisri rods* hwiifctH $5$ ixH atteelmtoft*.aswi «'ftfossy forato$ta&amp; m|w:tXiY*iy fl Hi4* e&amp;xut* kinase xifxfnu<r; 'ND4* tfrpert.ft'iem txd <fc«so ni>.S‘? me&amp;HS jR-iyjuymu-i "i'C^ ax^ffs poiyexrbs-xaUr f> f*&amp;'‘ ifrC" flxsasss· hk^'l of pelysfvfcfie asstS p»iyosrfxuuk:; ’OX?* ftxxifts cydk exfftt cejxrfymer:: ΧΧ*4" raea»* rerttwa piasrwa; x«:a«* ixxxx>>»ve pfasm» x il.ixx&amp;tf k* c&amp;X&amp; attack aftii gre« into cakmks that eax Ex' pessXiieri abv'tti .5· *&amp;»&amp;: \s1xh groxth rate nk'difre* yporjiajxxtuXyi ^ fcttrtw&amp;x £S ceEhtatl&amp;eh χίχΙ grv« im>>.c<>fc>s>m fhaf spox?»}X*js;:*fy dXfmxttisAc fceiarc the ttraf p&amp;ssagirtg **s .Human 5vS cd is uiuu&amp; i«ui ?.'idvv mix wktfsks nX fceskaf) avyjkjbte for iniaiytt*

Table .12: Surface Elemental Composition as Determined fey XFS

* Not plasma treated ,. ** Other dements were detected at a eanmtiraiion of 0.2-2,0%.

Table 13: Carbon Bonding Environment by CIs Spectra Carve Fitting

* Not plasma treated.

Table 14; Nitrogen Bonding Environment by Ms. Spectra Carve Fitting

* Not plasma treated. ** ND. analyzed, but not detected.

Claims (24)

1. A method to attach cells to a corona-plasma treated polystyrene surface containing at least 1.7% N, lacking a feeder cell layer and lacking an adlayer, comprising the steps of: a. Obtaining a suspension of the cells, and b. Adding the suspension of cells to the surface and allowing the cells to attach.

2. A method to attach cells to a surface lacking a feeder cell layer and lacking an adlayer, comprising the steps of: a. Obtaining a suspension of cells, and b. Adding the suspension of cells to the surface and allowing the cells to attach, wherein the surface has one of the following features: contains at least 1.7% N, has a sum of O and N of at least 29.6% and has a contact angle of at least 14.3 degrees; contains at least 2.0% N, has a sum of O and N of at least 30.7% and has a contact angle of at least 18.4 degrees; contains at least 2.1% N, has a sum of O and N of at least 30.2% and has a contact angle of at least 17.4 degrees; or contains at least 1.8% N, has a sum of O and N of at least 28.2% and has a contact angle of at least 18.8 degrees.

3. The method of claim 2, wherein the surface comprises corona plasma treated polystyrene.

4. The method of claim 1 or claim 3, wherein the corona plasma treated polystyrene surface contains at least 1.7% N, has a sum of O and N of at least 29.6% and has a contact angle of at least 14.3 degrees.

5. The method of claim 1 or claim 3, wherein the corona plasma treated polystyrene surface contains at least 2.0% N, has a sum of O and N of at least 30.7% and has a contact angle of at least 18.4 degrees.

6. The method of claim 1 or claim 3, wherein the corona plasma treated polystyrene surface contains at least 2.1% N, has a sum of O and N of at least 30.2% and has a contact angle of at least 17.4 degrees.

7. The method of claim 1 or claim 3, wherein the corona plasma treated polystyrene surface contains at least 1.8% N, has a sum of O and N of at least 28.2% and has a contact angle of at least 18.8 degrees.

8. The method of any one of claims 1 to 7, wherein the cells are maintained in culture after the cells attach to the surface.

9. The method of any one of claims 1 to 8, wherein the suspension of cells is a suspension of clusters of cells.

10. The method of any one of claims 1 to 8, wherein the suspension of cells is a suspension of single cells.

11. The method of any one of claims 1 to 10, wherein the cells are pluripotent stem cells.

12. The method of any one of claims 1 to 10, wherein the cells are human pluripotent stem cells.

13. The method of any one of claims 1 to 10, wherein the cells are human embryonic stem cells.

14. The method of any one of claims 1 to 13, further comprising treating the suspension with a compound capable of inhibiting Rho kinase activity prior to adding the suspension of cells to the surface.

15. The method of claim 14, wherein the compound capable of inhibiting Rho kinase activity is selected from the group consisting of: Y-27632, Fasudil, and Hydroxyfasudil.

16. The method of any one of claims 1 to 13, further comprising treating the suspension of cells with a compound capable of inhibiting Rho activity prior to adding the suspension of cells to the surface.

17. The method of claim 16, wherein the compound capable of inhibiting Rho activity is a Rho GTPase inhibitor or exoenzyme C3 Transferase.

18. The method of any one of claims 1 to 7, wherein the surface is part of a vessel or matrix.

19. A surface that is part of a vessel or matrix when used in cell culture or analysis, lacking a feeder cell layer and lacking an adlayer, wherein the surface allows the attachment and cultivation of cells, and wherein the surface has one of the following features: contains at least 1.7% N, has a sum of O and N of at least 29.6% and has a contact angle of at least 14.3 degrees; contains at least 2.0% N, has a sum of O and N of at least 30.7% and has a contact angle of at least 18.4 degrees; contains at least 2.1% N, has a sum of O and N of at least 30.2% and has a contact angle of at least 17.4 degrees; or contains at least 1.8% N, has a sum of O and N of at least 28.2% and has a contact angle of at least 18.8 degrees.

20. The surface of claim 19, wherein the surface comprises corona plasma treated polystyrene.

21. The surface of claim 19 or claim 20, wherein the surface contains at least 1.7% N, has a sum of O and N of at least 29.6% and has a contact angle of at least 14.3 degrees.

22. The surface of claim 19 or claim 20, wherein the surface contains at least 2.0% N, has a sum of O and N of at least 30.7% and has a contact angle of at least 18.4 degrees.

23. The surface of claim 19 or claim 20, wherein the surface contains at least 2.1% N, has a sum of O and N of at least 30.2% and has a contact angle of at least 17.4 degrees.

24. The surface of claim 19 or claim 20, wherein the corona plasma treated polystyrene surface contains at least 1.8% N, has a sum of O and N of at least 28.2% and has a contact angle of at least 18.8 degrees.