JP6145324B2 - Cell culture substrate that promotes differentiation into nerve cells - Google Patents

Description

  The present invention relates to a cell culture substrate that promotes differentiation induction from neural stem cells into nerve cells, and a differentiation induction method using the cell culture substrate.

  With the development of regenerative medicine technology using cells, the development of technology for inducing differentiation into the target cells has been actively conducted. In particular, it is essential to establish a technique for inducing differentiation from a stem cell to a target cell in a short period of time. Among various cell types, nerve cells are known to hardly regenerate spontaneously except for some parts when they become adults, and therefore, studies on differentiation induction thereof are underway.

  Induction of differentiation of nerve cells in a culture system is performed by adding various humoral factors to the medium, and there are various types of humoral factors. For example, Sonic hedgehog (Shh) is expressed at an early stage of development and is known to induce differentiation specifically in the nervous system by forming a concentration gradient in the body (Non-patent Document 1). It is also known that Shh has an effect of promoting differentiation from cultured neural stem cells to neural cells when added to a medium (Non-patent Document 2). Furthermore, it has also been reported that axonal elongation is observed during culture by adding Shh (Non-patent Documents 3 and 4).

  Thus, a method of adding a humoral factor is generally used for promoting differentiation induction. Although these humoral factors are expensive, in order to continuously send a differentiation-inducing signal to the cells during culture, it is necessary to administer each time the medium is changed. Conventional methods of adding humoral factors to the medium Is disadvantageous in terms of cost.

  Therefore, in Non-patent Document 5, differentiation induction from mouse ES cells to motor neurons is promoted by coculturing with genetically modified cells that produce Shh and always supplying Shh. However, the above method takes time and effort to prepare genetically modified cells. Moreover, there is a problem that differentiation induction efficiency is poor because the probability that the humoral factor comes into contact with the cultured cells is low only by suspending the humoral factor in the liquid.

The EMBO Journal (28) 457-465 (2009) The Journal of Neuroscience (23) 9862-9872 (2003) Development (128) 3927-3936 (2001) Cell (113) 11-23 (2003) Stem Cells (25) 1697-1706 (2007)

  An object of the present invention is to provide means for inducing differentiation of neural cells from neural stem cells efficiently and at low cost.

  By culturing neural stem cells using a cell culture substrate obtained by covalently immobilizing a protein having an activity of inducing differentiation of neural stem cells into neural cells on a support, an efficient and low cost method is provided. It was found that neuronal cells can be induced to differentiate.

That is, the present invention includes the following.
(1) A cell culture substrate for inducing differentiation of neural stem cells into neural cells,
A support having an active group on the surface, and a protein having an activity of inducing differentiation of neural stem cells into nerve cells, which is immobilized on the support through a covalent bond by reaction with an active group on the surface of the support,
The cell culture substrate comprising:
(2) The cell culture substrate according to (1), wherein a protein having an activity of inducing differentiation of neural stem cells into neural cells has a function of inducing differentiation without being incorporated into the cells.
(3) The cell culture substrate according to (2), wherein the protein having an activity of inducing differentiation of neural stem cells into neural cells is Shh.
(4) The cell culture substrate according to any one of (1) to (3), wherein the active group is a carboxyl group or an active ester group and is present on a hydrophilic polymer bonded to the support surface.
(5) A method for inducing differentiation of neural stem cells into neural cells, comprising the step of culturing neural stem cells on the cell culture substrate according to any one of (1) to (4).

  According to the present invention, differentiation induction from a neural stem cell to a neural cell and axonal elongation can be promoted efficiently and at low cost.

It is the schematic which shows one Embodiment of this invention. It is the schematic which shows one Embodiment of this invention. It is a photograph which shows the result of having imaged the signal by the antigen antibody reaction of the cell culture substrate by which Shh was fix | immobilized with the CCD imager. It is a graph which shows the average value of the signal strength by the antigen antibody reaction of the cell culture substrate which fix | immobilized Shh with the aqueous solution of various density | concentrations. It is a photograph which shows the result of having imaged the thing which culture | cultivated the neural stem cell on the cell culture base material, and immunostained the obtained cell with a confocal microscope. It is a graph which shows the result of having immunostained the cell obtained by culture | cultivating a neural stem cell on a cell culture base material, and having calculated the ratio of the Tuj-1 positive nerve cell with respect to the number of DAPI positive cells. It is the schematic of a nerve cell. It is a graph which shows the result of having measured the axon length about Tuj-1 positive cell among the cells obtained by culturing a neural stem cell on a cell culture substrate.

  The support used for the cell culture substrate of the present invention is not particularly limited as long as it can provide a stable surface against water. Specific materials constituting the support include inorganic materials such as metal, metal oxide, glass, quartz, silicon, and ceramic, elastomer, plastic, polyester resin, polyethylene resin, polystyrene resin, polypropylene resin, ABS resin, nylon And synthetic polymers such as acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin, and natural polymers such as chitin, chitosan, and cellulose. . The shape of the support is not limited, and examples thereof include flat shapes such as flat plates, flat membranes, films, and porous membranes, and three-dimensional shapes such as cylinders, stamps, multiwell plates, microchannels, and fine particles. . In particular, a support made of glass, quartz or silicon is desirable.

  The active group on the surface of the support is not particularly limited as long as it can form a covalent bond with a functional group on the protein, but is preferably a carboxyl group and an active ester group. On the surface of the support, both carboxyl groups and active ester groups may be present as active groups, or only one of them may be present. The active group is preferably present on a hydrophilic polymer bound to the support surface. In other words, it is bonded to the support surface via a hydrophilic polymer chain.

  The method for introducing the active group onto the surface of the support is not particularly limited, and can be carried out, for example, by the methods described in JP2009-156864A and JP2013-11480A. Specifically, a step of introducing a functional group onto the surface of the support, a step of bonding a hydrophilic polymer having a binding group capable of binding to the functional group and a hydroxyl group to the functional group, and a step on the bonded hydrophilic polymer. In the step of forming a carboxyl group by subjecting the hydroxyl group to a ring-opening half esterification reaction with a cyclic acid anhydride, the carboxyl group can be bonded to the support surface via a hydrophilic polymer. Furthermore, by converting the formed carboxyl group into an active ester, the active ester group can be bound to the support surface via a hydrophilic polymer.

  The functional group introduced to the surface of the support is not particularly limited as long as it can be covalently bonded to the binding group on the hydrophilic polymer. However, when the support is glass, quartz or silicon, it is desirable that the support is an epoxy group, an aldehyde group or an amino group which can be easily introduced with a general-purpose silane coupling agent. In addition, an N-hydroxysuccinimide group, a hydroxyl group, an isocyanate group, a maleimide group, a thiol group, a carboxyl group, a carbodiimide group, and the like are conceivable, but not limited thereto. General-purpose silane coupling agents that can be used in the present invention include silane coupling agents having an epoxy group, an aldehyde group, or an amino group. Specifically, 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropyltriethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxy Propyldimethylethoxysilane, 2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane, 2- (3,4-epoxycyclohexyl) ethyltriethoxysilane, N- (2-aminoethyl) -3-aminopropyltrimethoxy Silane, N- (2-aminoethyl) -3-aminopropylmethyldimethoxysilane, N- (2-aminoethyl) -3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxy Silane, 3-aminopropylmethyl Silane, 3-aminopropyl methyl diethoxy silane and 3-aminopropyl dimethyl ethoxy silane.

  After introducing an epoxy group to the support surface, it can be hydrolyzed to a diol and then converted to an aldehyde group by oxidative cleavage. An aldehyde group can also be introduced by reacting a compound having two aldehyde groups with the introduced amino group (for example, glutaraldehyde).

  Examples of the application method of the general-purpose silane coupling agent to the support surface include, but are not limited to, an immersion method, a sol-gel method, a vapor deposition method, a spray method, and an integral blend method. A desirable method is the sol-gel method.

  The hydrophilic polymer having a binding group and a hydroxyl group functions as a hydrophilic spacer interposed between the support and the protein. It is particularly preferable that the hydrophilic polymer has a binding group at one end and a hydroxyl group at the other end. The binding group may be a hydroxyl group or a functional group different from the hydroxyl group (for example, amino group, epoxy group, aldehyde group, carboxyl group, N-hydroxysuccinimide group, isocyanate group, maleimide group, A thiol group, a carbodiimide group, etc.), but a hydroxyl group is preferred.

  The hydrophilic polymer is preferably at least one selected from the group consisting of ethylene glycol, a polymer of ethylene glycol, and a copolymer of ethylene glycol and propylene glycol. The polymer of ethylene glycol is not particularly limited as long as it is a polymer in which two or more molecules of ethylene glycol are polymerized, for example, a number average molecular weight of 176 or more, but a number average molecular weight of 1,000 or more is preferable, and a number average molecular weight of 10,000 or less. Preferred is 4000 or less. The copolymer of ethylene glycol and propylene glycol is preferably a block copolymer, and in particular, at least one unit of ethylene glycol unit at each end of a (poly) propylene glycol block composed of one or more units of propylene glycol unit. A block copolymer obtained by copolymerization of a (poly) ethylene glycol block consisting of is preferable. The number average molecular weight of the copolymer of ethylene glycol and propylene glycol is not particularly limited as long as it is 1000 or more, but the number average molecular weight is preferably 12,000 or less, and more preferably 4000 or less. In the present invention, propylene glycol refers to 1,2-propanediol. As the hydrophilic polymer, a phosphocholine-like polymer (MPC polymer) as described in JP-A-2002-356519 may be bound to the support surface.

  When a hydrophilic polymer having two hydroxyl groups is bonded to the epoxy group introduced on the support surface, one hydrophilic compound is bonded to one epoxy group and introduced on the support surface. When a hydrophilic polymer having two hydroxyl groups is bonded to an aldehyde group, two hydrophilic polymers are bonded to one aldehyde group.

  A cyclic acid anhydride is subjected to a ring-opening half-esterification reaction with a hydroxyl group on the hydrophilic polymer (in the case where the binding group is a hydroxyl group, the hydroxyl group not subjected to the above reaction). A carboxyl group derived from an anhydride can be formed. In terms of production cost, the cyclic acid anhydride is preferably succinic anhydride or glutaric anhydride, but is not limited thereto. The ring-opening half esterification reaction is preferably performed in an inert organic solvent such as toluene to which a catalyst is added. Examples of the catalyst used in the ring-opening half esterification reaction include triethylamine, isobutylethylamine, pyridine, 4-dimethylaminopyridine and the like.

  Active esterification of the carboxyl group is not essential. However, since the active ester is more reactive than the carboxyl group, the active esterification is preferably performed when it is desired to quickly immobilize the protein.

  The active ester serves to bind the hydrophilic polymer and protein covalently. Here, the active ester means a chemical structure of R—C (═O) —X. X represents a leaving group such as halogen, N-hydroxysuccinimide group or derivatives thereof, 1-hydroxybenzotriazole group or derivatives thereof, pentafluorophenyl group, paranitrophenyl group, but is not limited thereto. As the active ester, N-hydroxysuccinimide ester is desirable in terms of reactivity, safety and production cost. Conversion of the carboxyl group to N-hydroxysuccinimide ester is achieved by reacting the carboxyl group with N-hydroxysuccinimide and carbodiimide simultaneously. Here, carbodiimide means an organic compound having a chemical structure of -N = C = N-, for example, dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, etc. Although considered, it is not limited to these. The concentration of N-hydroxysuccinimide and carbodiimide is preferably set in the range of 1 to 100 mM, the reaction temperature is 4 to 100 ° C., and the reaction time is in the range of 2 minutes to 16 hours. As the reaction solvent, N, N'-dimethylformamide (DMF), toluene or the like can be used.

  The protein is preferably immobilized on the support by reacting its amino group with an active group on the support surface, preferably a carboxyl group or an active ester to form a covalent bond. The reaction between the active ester on the support and the protein is performed, for example, as follows. First, citrate buffer (pH 3.0 to 6.2), acetate buffer (pH 3.6 to 5.6), phosphate buffered saline (PBS, pH 5.8 to 8.0), carbonate-heavy An aqueous protein solution of 0.1 μg / ml to 1 mg / ml is prepared using a buffer solution that does not contain an amino group component, such as carbonate buffer (pH 9.2 to 10.6). It is desirable to optimize the buffer or pH so as to maximize the amount of protein immobilized. The buffer may contain stabilizers such as glycerol and polyethylene glycol, salts (NaCl), surfactants and the like, and these do not inhibit the reaction between the active ester and the protein. When this aqueous solution is brought into contact with the active ester surface, a functional group such as an amino group of the protein reacts with the active ester to form an amide bond. As a result, the protein is immobilized on the support surface by a covalent bond. Here, the reaction temperature may be set in the range of 4 to 37 ° C., and the contact time in the range of 2 minutes to 16 hours. After immobilizing the protein, it is desirable to wash the carrier with an appropriate washing solution. At this time, the washing solution is preferably a buffer solution containing about 0.5M salt (NaCl) and about 0.1% nonionic surfactant. As a result, it is possible to remove proteins that are only physically adsorbed without being covalently bonded.

In one embodiment, the cell culture substrate obtained in this way is,
A support having a functional group on the surface;
A hydrophilic polymer having a binding group capable of binding to the functional group and a hydroxyl group, and a carboxylic acid compound having two carboxyl groups,
A bond is formed between the functional group on the support and the binding group of the hydrophilic polymer;
An ester bond is formed between one carboxylic acid group of the carboxylic acid compound and a hydroxyl group of the hydrophilic polymer, and the other carboxyl group is free or active esterified;
A protein having an activity of inducing differentiation of neural stem cells into nerve cells is immobilized on a support through a covalent bond by reaction with a carboxyl group or an active ester group.

  In the present invention, the protein immobilized on the cell culture substrate is a protein having an activity of inducing differentiation of neural stem cells into neural cells. A protein having a function of inducing differentiation of neural stem cells into neurons without being taken up by cells is preferred. In other words, a protein having a function of inducing differentiation by signal transduction through a receptor without being taken up into cells is preferable. By immobilizing a protein that has the function of inducing differentiation of neural stem cells into neurons without being taken up by the cells, the protein is not consumed in differentiation induction but remains on the cell culture substrate, allowing it to function repeatedly. It is not necessary to add every time. Therefore, it is advantageous from the viewpoint of cost and operability. In addition, since the cells to be cultured are adhered to the cell culture substrate on which the protein is immobilized, the cells are more likely to come into contact with the protein, and it is thought that the differentiation induction of nerve cells and the axon elongation are further promoted. It is done. On the other hand, when protein is added to the medium, it is considered that the probability of contact with cells is low and the differentiation rate is slow because it is floating in the medium (FIG. 1). For example, as shown in FIG. 2, since the substrate and the protein are immobilized by a covalent bond, the possibility that the protein is peeled off from the surface is smaller than in the case of physical adsorption. Therefore, the protein immobilization amount can be kept constant over the culture period. Therefore, differentiation induction can be promoted stably.

  Proteins included in morphogens expressed during development such as Shh, FGF, BMP, Wnt, Notch, EGF, activin, etc., as proteins having the function of inducing differentiation by signal transduction through receptors without being taken up into cells, and , NGF, BDNF, NT-3, NT-4, NT-5, PDGF, GDNF, CNTF, LIF, TGF, artemin, nurturin, and parsephin, preferably Shh is immobilized. Shh is the most important morphogen in development and plays a role in designing many organ systems such as the extremities and the cerebrospinal midline structure. It has been reported in, for example, Nature Reviews Neuroscience (3) 24-33 (2003) that Shh has a function of inducing differentiation through signal transduction through a receptor.

  The origin of the protein may be any of mammals, birds, fishes, reptiles, and amphibians, and is not particularly limited, but is preferably a mammal (for example, a rodent such as a mouse or a primate such as a human), particularly preferably. Is mouse or human. It is preferable to use those derived from the same animal as the nerve cells that induce differentiation. In order to evaluate whether the target protein is immobilized on the surface, immunostaining using an antibody against the target protein can be used.

  The amount of protein to be immobilized is appropriately determined depending on the type of protein, but when immobilizing Shh, it preferably has an active group on its surface in a Shh aqueous solution having a concentration of 0.5 to 4.9 μg / ml. By immersing the support, Shh is immobilized to the support through a covalent bond. By using the Shh aqueous solution in the above concentration range, it is possible to induce differentiation of nerve cells effectively.

  After the protein is immobilized (preferably after further washing), it is less reactive by binding an unreacted active group, preferably a carboxyl group or an active ester group, to a low molecular weight compound having an amino group. It is preferably converted into a functional group and inactivated (also called blocking). This can prevent unintended molecules from being improperly immobilized. This operation is particularly necessary when the active group is an active ester.

  It is desirable that the surface of the cell culture substrate after reacting with the low molecular weight compound is hydrophilic. This is because a hydrophilic surface generally has an effect of suppressing nonspecific adsorption of biological substances such as proteins. For this purpose, it is preferable to use a low molecular compound having an amino group and further having a hydrophilic group in addition to the amino group. Non-limiting examples of such low molecular weight compounds include ethanolamine, trishydroxymethylaminomethane, diglycolamine (IUPAC name: 2- (2-aminoethoxy) ethanol). Especially, diglycolamine is excellent in the effect which suppresses nonspecific adsorption | suction of a biological substance. These low molecular weight compounds are dissolved in a buffer solution such as PBS so as to be 10 to 50 mM, and this is brought into contact with a carrier on which a desired substance is immobilized. The reaction temperature may be set in the range of 4 to 37 ° C. and the reaction time in the range of 2 minutes to 16 hours.

The cell culture substrate used in the present invention is preferably precoated before the inactivation (blocking) for the purpose of promoting cell adhesion. The precoat treatment is derived from a living product such as an extracellular matrix (collagen, fibronectin, proteoglycan, laminin, vitronectin), gelatin, poly-L-ornithine, poly-L-lysine, adhesamine, CellStart ^™, or animal serum. It can be carried out by coating the cell culture substrate with a material or the like. By performing the precoat treatment, adhesion of stem cells with low adhesion can be promoted, and cell adhesion culture and differentiation induction can be effectively performed. Although there is no restriction | limiting in the coating method, the coating by immersion is common. By culturing stem cells by adhering them to the cell culture substrate surface, the stem cells can be brought into contact with the protein on the substrate surface and differentiation can be efficiently induced.

  Examples of the neural cell induced to differentiate according to the present invention include a neural cell, a neural tube cell, a neural crest cell, and the like. A nerve cell refers to a cell having a function of transmitting a stimulus to another nerve cell, a muscle, or a gland cell upon receiving a stimulus from another nerve cell or a stimulus receptor cell. Nerve cells can be classified according to differences in neurotransmitters produced by the neurons, for example, according to differences in secreted neurotransmitters. Examples of nerve cells classified by these neurotransmitters include dopamine secreting neurons, acetylcholine secreting neurons, serotonin secreting neurons, noradrenaline secreting neurons, adrenergic secreting neurons, glutamate secreting neurons, and the like.

  In another aspect, nerve cells can be classified according to the difference in site where the nerve cells are present. Examples of the nerve cells classified at these sites include forebrain neurons, midbrain neurons, cerebellar neurons, hindbrain neurons, spinal cord neurons, and the like.

  The origin of the nerve cell may be any of mammals, birds, fishes, reptiles and amphibians, and is not particularly limited, but is preferably a mammal (for example, a rodent such as a mouse or a primate such as a human), Preferred is mouse or human.

  Neural stem cells used for differentiation induction are cells that have the ability to differentiate into neurons, astrocytes, and oligodendrocytes, and have the ability to self-replicate. It has a function to supply. As a method for confirming that it is a neural stem cell, a method for actually transplanting it to the brain and confirming its differentiation ability, a method for confirming differentiation of neural stem cells into neural cells, astrocytes and oligodendrocytes in vitro, etc. (Mol. Cell. Neuroscience, 8, 389 (1997); Science, 283, 534 (1999)). In addition, neural stem cells having such functions can be stained with an anti-nestin antibody that recognizes the cytoskeletal protein nestin, a marker that has been confirmed to be expressed in neural progenitor cells (Science, 276, 66 (1997). ). Therefore, neural stem cells can be confirmed by staining with an anti-nestin antibody.

  In order to culture neural stem cells on a cell culture substrate, not only seeding the neural stem cells on the substrate surface of the present invention and starting the culture, but also embryonic stem cells, induced pluripotent stem cells, mesenchymal systems This includes culturing stem cells, such as stem cells, that are more pluripotent than neural stem cells on the cell culture substrate of the present invention, differentiation into neural stem cells, and continuing to culture these cells. It is desirable to start culturing by seeding on the surface.

  Various stem cells are known and their preparation methods are also known. In addition, stem cell lines of various animals have been established. A person skilled in the art can appropriately obtain various stem cells. For example, it is known that neural stem cells exist in brain and spinal cord tissues, and neural stem cells can be collected from these tissues. In addition, neural stem cells can be obtained from embryonic stem cells, mesenchymal stem cells, and induced pluripotent stem cells (Mol Cell Neurosci. 22 (4) 501-515 (2003); Stem Cell Rev and Rep, 6, 270- 281 (2010); PNAS, 108 (19) 7838-7843 (2011)). Neural stem cells prepared by reprogramming such as gene transfer, established neural stem cells, or neural stem cells obtained from animals can also be used (Cell Stem Cells, (11) 100-109 (2012)).

  Neural stem cells before seeding on a cell culture substrate are maintained undifferentiated using a non-differentiation-inducing medium. Before and after seeding on the cell culture substrate surface, the medium is switched to the differentiation-inducing medium, seeded on the substrate surface, and the cells are allowed to grow until they become confluent.

The medium used for inducing differentiation of nerve cells on the cell culture substrate of the present invention may be the same as a conventionally known medium. Various media for differentiation induction culture of nerve cells are known. For example, [3 μM Glutamax, 5 μg / ml heparin and 100 unit / mL penicillin, 100 μg / mL streptomycin-containing DMEM / F12 (1: 1), added Factor: 2% B27, 1% N ₂ ] is widely used as the basic composition. B27 and N2 are medium additives for culturing nerve cells, and are used in place of serum when culturing nerve cells. Various other basic compositions are also known. A substance useful for inducing differentiation of a nerve cell (a substance other than a protein immobilized on a cell culture substrate) may be appropriately added to a medium having such a basic composition. For example, retinoic acid is often used in inducing differentiation into nerve cells, and is usually added to the medium at a concentration of about 1 μM.

The seeding density of the neural stem cells on the cell culture substrate is not particularly limited as long as it follows a conventional method. A density of less than 1.5 × 10 ⁵ cells / cm ^{2 with} respect to the cell culture substrate, preferably a density of 1.0 × 10 ⁵ cells / cm ² or less, preferably a density of 3 × 10 ⁴ cells / cm ² or more. Sow. Thereby, the differentiation promoting effect by the paracrine effect between cells is anticipated, and inhibition of cell adhesion and differentiation induction due to excessive cell concentration can be suppressed.

The culture temperature is usually 37 ° C. It is preferable to culture in a CO ₂ concentration atmosphere of about 5% using a CO ₂ cell culture device or the like.

  The culture period after seeding the neural stem cells on the cell culture substrate is preferably 4 days or more, more preferably 7 days or more. By allowing this period, neuronal differentiation can be sufficiently induced.

  Neuronal cells that have been induced to differentiate on the cell culture substrate of the present invention can be recovered by pipetting, physical detachment with a cell scraper, or by enzymatic treatment (trypsin, dispase, etc.) and used for a desired application. .

  When dopaminergic cells are obtained as nerve cells according to the present invention, they can be used for treatment of Parkinson's disease by transplanting them with a catheter or the like. For example, when oligodendrocyte progenitor cells are obtained, they can be used for treatment of spinal cord injury by direct injection into the affected area or transplantation using a catheter or the like. Further, for example, when neuron precursor cells or astrocyte precursor cells are obtained, they can be used for the treatment of motor neuropathy by directly injecting them into the affected area or transplanting them using a catheter or the like. The nerve cells obtained by the present invention can be used as a drug discovery research tool by being used for evaluating the efficacy / toxicity of a test substance, elucidating an action mechanism, or analyzing a biological phenomenon mechanism.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the scope of the examples.

<Example 1>
39 g of toluene (Pure Chemical), 450 μl of 3-glycidoxypropyltrimethoxysilane (TSL8350, GE Toshiba Silicone) and 900 μl of triethylamine (Wako Pure Chemical Industries, Ltd.) are mixed, and this is a UV cleaned 10 cm square glass substrate. (Corresponding to the support) was immersed and gently shaken at room temperature for 20 hours. Thereafter, the substrate was washed with ethanol and water and dried by nitrogen blowing. This operation introduced epoxy groups on the glass surface. Next, the substrate was immersed in tetraethylene glycol (TEG, Kanto Chemical) containing a catalytic amount of concentrated sulfuric acid and heated at 80 ° C. for 1 hour. After the reaction, the substrate was thoroughly washed with water and dried by nitrogen blowing. By this operation, TEG reacted with the epoxy group to form a covalent bond. Next, 100 mg of succinic anhydride (SuA, Kanto Chemical) and 120 mg of 4-dimethylaminopyridine (DMAP, Wako Pure Chemical Industries) were dissolved in 43 g of toluene, and the substrate was immersed therein at 80 ° C. for 1 hour. Heated. Thereafter, the substrate was washed with ethanol and water and dried by nitrogen blowing. By this operation, the ring-opening half esterification reaction proceeded, and a carboxyl group was introduced at the free end of the TEG. Next, 290 mg of N-hydroxysuccinimide (NHS, Wako Pure Chemical Industries) and 390 μl of N, N′-diisopropylcarbodiimide (DIC, Wako Pure Chemical Industries) were added to 47 g of N, N′-dimethylformamide (DMF, Kanto Chemical). It melt | dissolved, the said board | substrate was immersed here, and it heated at 80 degreeC for 1 hour. Thereafter, the substrate was washed with ethanol and water and dried by nitrogen blowing. By this operation, an N-hydroxysuccinimide group (NHS group) was introduced at the free end of the TEG. Finally, the substrate was cut into a size of 25 mm × 10 mm, sterilized with 70% ethanol, washed with PBS, and placed on the bottom of the dish.

  A solid phase buffer was prepared by adding 0.6 M sodium chloride (Wako Pure Chemical Industries, Ltd.) and 5% glycerin (Wako Pure Chemical Industries, Ltd.) by volume to 0.05 M carbonate buffer (pH 9.6). To this, Shh (R & D Systems) was diluted to a concentration of 10 μg / ml. Each 1 ml of this was placed in the dish and shaken to spread and left in a clean bench for 5 minutes. Thereafter, the solution was collected with a pipette and dried for 30 minutes to immobilize Shh on the substrate surface. Blocking operation was performed for 1 minute with PBS containing diglycolamine. After washing with PBS, a rabbit IgG-labeled anti-Shh antibody diluted with PBS (Millipore dilution ratio 1/2000) was added and allowed to react at room temperature for 1 hour. After washing 3 times with PBS, the reaction was carried out for 30 minutes at room temperature using an HRP-labeled anti-rabbit IgG antibody (dilution rate 1/2000).

  The substrate was taken out from the dish, and light was emitted by Immunostar LD (Wako Pure Chemical Industries, Ltd.), and then an image was obtained with a CCD imager LAS4000 (GE Healthcare). The intensity of the emission intensity was measured by designating a region in the substrate surface. The results are as shown in FIG. 3. A signal due to the antigen-antibody reaction of Shh was observed, and it was found that Shh was immobilized on the entire surface of the substrate.

<Example 2>
In order to examine the correlation between the amount of Shh immobilized on the surface and the signal intensity, the following analysis was performed.
Dilute Shh to a concentration of 0, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50 μg / ml with the immobilization buffer described in Example 1. Using a pipette (Gilson), spotting was performed in two rows vertically on the substrate into which the active ester group prepared in Example 1 was introduced.

  Shh was immobilized on the surface by drying at room temperature for 5 minutes. After washing with PBS, blocking operation, PBS washing, and Shh detection using an antibody were performed in the same manner as in the method described in Example 1. Note that the emission intensity of the signal spot was calculated by calculating the in-plane intensity of each spot using the software (ImageQuant LAS4000) attached to the LAS4000, and the average value of the emission intensity was obtained. The results are shown in FIG. From this, it was shown that there is a correlation between the concentration of the Shh solution used and the amount of surface immobilization.

<Test Example 1>
The differentiation induction efficiency of neural stem cells was examined using a substrate on which Shh was immobilized on the surface and a substrate on which Shh was not immobilized. The following analysis was performed using neural stem cells derived from human ES cells (Life Technologies) in accordance with the attached manual. Details will be described below.

  Knockout DMEM / containing a basic FGF, EGF and StemPro (registered trademark) by seeding neural stem cells derived from human ES cells on a 6 cm polystyrene dish (Asahi Glass Co., Ltd.) coated with poly-L-ornithine and laminin (Becton Dickinson). Grow in F-12 medium (all from Life Technologies). In addition, medium exchange was performed about the half quantity by the frequency once every 2-3 days. The undifferentiated nature of these neural stem cells is determined by immunostaining using a rabbit IgG-labeled anti-nestin antibody (Sigma dilution ratio 1/500) and a rabbit IgG-labeled anti-SOX2 antibody (Millipore dilution ratio 1/500). And mouse IgG1-labeled anti-Tuj-1 antibody (Promega dilution ratio 1/500), which is a marker for neurons, was used for confirmation. It was confirmed that nestin and SOX2 were positive and Tuj-1 was negative. From this, it was confirmed that the cells used were neural stem cells.

  In the same manner as in Example 1, a Shh solution having a concentration of 0, 0.05, 0.1, 0.5, 1 μg / ml was spotted on a substrate into which an active ester group had been introduced, thereby preparing a Shh-immobilized substrate. Then, after coating the substrate with poly-L-ornithine (concentration 1 μg / ml; Sigma) and laminin (concentration 50 μg / ml; Becton Dickinson) diluted with PBS, blocking with a diglycolamine solution was similarly performed. went.

  In Comparative Example 1, a normal 3.5 cm polystyrene dish (Asahi Glass Co., Ltd.) was used that was similarly coated with poly-L-ornithine and laminin on the bottom surface. In Comparative Example 2, the bottom surface was coated for 30 minutes by immersing PBS containing Shh at a concentration of 1 μg / ml in a normal 3.5 cm polystyrene dish (Asahi Glass Co., Ltd.), washed with PBS, and similarly poly-L- The bottom coating was used with ornithine and laminin. In Comparative Example 3, a polystyrene dish with a bottom coating was used as in Comparative Example 1, and Shh was added to the medium at a concentration of 1 μg / ml. In Comparative Example 3, Shh was added to the medium at a concentration of 1 μg / ml each time the medium was changed.

The neural stem cells maintained as described above were allowed to react with Accutase (Life Technologies) at 37 ° C. for 7 minutes, and the cells were detached and collected by centrifugation at 1000 rpm for 5 minutes. 5 × 10 ⁵ neural stem cells were seeded on a Shh-immobilized substrate placed in a 3.5 cm dish and differentiated into neurons. Differentiation into nerve cells was carried out by culturing in Knockout DMEM / F-12 medium (all Life Technologies) containing B27 trophic factor and Glutamax-I for 1 week. In addition, medium exchange was performed about the half quantity by the frequency once every 2-3 days. After 1 week, immunostaining was performed by the following method for evaluation.

  After washing with PBS, a 4% paraformaldehyde solution was added, and the cells were fixed by allowing to stand at room temperature for 20 minutes. Further, after washing with PBS, blocking is performed for 30 minutes or more at room temperature with a PBS solution containing Triton X-100 (Nacalai Tesque) at a volume ratio of 0.1% and Normal Goat Serum (NGS; DAKO) at a volume ratio of 3%. It was. Subsequently, the primary antibody solution diluted in the same blocking solution was incubated overnight at 4 ° C. After washing three times with PBS, staining was performed by incubating with a secondary antibody solution diluted in PBS for 1 hour at room temperature. After the reaction, it was washed 3 times with PBS.

  The following antibodies were used for staining. Mouse IgG1-labeled anti-Tuj-1 antibody (dilution ratio 1/500), which is a marker of neural stem cells, as the primary antibody, Alexa488-labeled mouse IgG1 antibody (Molecular Probes; dilution ratio 1/1000) diluted in PBS as the secondary antibody ) Was used. Cell nuclei were stained by incubating at room temperature for 10 minutes with 4 ′, 6-diamidino-2-phenylindole (DAPI; Sigma) diluted 1/1000 with PBS.

  After staining, the image was captured with a confocal microscope (Zeiss) after encapsulating with a cover glass and Immunomount (DAKO). The ratio of Tuj-1 positive neurons to the number of DAPI positive cells was calculated. FIG. 5 shows an image obtained by the confocal microscope. Moreover, the result of having summarized the ratio of differentiation is shown in FIG. From this, Shh is immobilized on the substrate by covalent bond, so that Shh is simply added to the medium (Comparative Example 3) or Shh is physically adsorbed on the substrate (Comparative Example 2). It was found that neural differentiation is promoted. That is, it was shown that the present invention can effectively promote differentiation induction into nerve cells.

<Test Example 2>
Using the image diagram obtained in Test Example 1, the length of the axon was estimated. The details are described below.
For Tuj-1 positive cells, the length of the axon from the cell body was measured by ImageJ (NIH), and an average value was obtained and compared. In addition, as shown in FIG. 7, the length measured with the longest axon from the cell body is defined as “axon length” for convenience.

  The measuring method using ImageJ is as follows. First, an image file for measurement is opened, and the actual length is made to correspond to the number of pixels on the screen by Set Scale. Next, the axon is traced from the cell body to the end by Freehand Selections. Thereafter, after measuring the length by Measurement, the straight line portion at the start and end is set by Straight line Selection, and the length is similarly measured by Measurement. The axon length is obtained by subtracting the latter length from the former length.

  The results are shown in FIG. From this result, by using a substrate on which Shh was immobilized by covalent bond, axonal elongation was observed as compared with Comparative Examples 1 and 2, and almost the same as the case of adding Shh of Comparative Example 3 to the medium. Axon outgrowth was observed. In Comparative Example 3, since Shh is added every time the medium is replaced, more Shh is used than in the case of immobilizing Shh. However, according to the present invention, an equivalent axon extension effect can be obtained while suppressing the amount of Shh used. It was shown that.

  It is considered that the required amount of Shh required for induction of differentiation into nerve cells and axonal elongation is different. Regarding the induction of differentiation of nerve cells, as shown in FIG. 6, when Shh is immobilized on a substrate, it is considered that concentration dependence is high because a small amount of immobilization is not effective. Therefore, in the method in which Shh is suspended in the culture medium, it is considered that differentiation does not proceed due to a decrease in the amount of Shh in contact with cells. Therefore, it is considered that the embodiment in which the Shh and the cell are always in contact as in the present invention is superior to the method of adding Shh to the medium. On the other hand, with respect to axon elongation, as shown in FIG. 8, when Shh is immobilized on a substrate, an effect is observed even with a low amount of immobilization, and it is considered that concentration dependence is low. Therefore, it is considered that the effect of axonal elongation caused by accidental binding of floating Shh to differentiated nerve cells is observed.

Claims (5)

A cell culture substrate for inducing differentiation of neural stem cells into neural cells,
A support having an active group on its surface; and
0.5~4.9μg / ml concentration of Shh solution is immobilized to the support by a covalent bond by reaction with active groups of the support surface in a Shh,
The cell culture substrate comprising: Active group is a carboxyl group or an active ester group, is present on a hydrophilic polymer bonded to the surface of the support, according to claim 1 cell culture substrate according. A method for inducing differentiation of neural stem cells into neural cells, comprising the step of culturing neural stem cells on the cell culture substrate according to claim 1 or 2 . A method for producing a cell culture substrate for inducing differentiation of neural stem cells into neural cells,
A method comprising immobilizing Shh covalently on a support by immersing the support having an active group on the surface thereof in an aqueous solution of Shh having a concentration of 0.5 to 4.9 μg / ml. The method according to claim 4, wherein the active group is a carboxyl group or an active ester group and is present on a hydrophilic polymer bound to the support surface.

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