JP7759765B2 - Spheroids and methods for producing same - Google Patents

Description

The present invention relates to spheroids and methods for producing them.

In recent years, attention has been focused on the creation of tissues that mimic the structure of organs by culturing cells in three dimensions. For example, nervous system spheroids, which contain nervous system cells and vascular system cells, have a structure similar to brain tissue, and are therefore expected to be useful in elucidating the pathological mechanisms of Alzheimer's disease or brain diseases caused by vascular disorders, as well as for drug screening in the field of drug discovery. For example, Non-Patent Document 1 reports an attempt to create brain-like spheroids by fusing spheroids made of neural progenitor cells derived from induced pluripotent stem cells (iPS cells) with spheroids made of vascular endothelial cells.

L. Song et al., Sci. Rep. 9, 5977(2019)

In conventional nervous system spheroids containing nervous system cells and vascular system cells, the vascular structure was not formed in a reticular pattern. The present invention was made in consideration of this issue, and aims to provide nervous system spheroids with a reticular vascular structure.

A spheroid according to one aspect of the present invention comprises nerve cells or glial cells, stem cells, and vascular endothelial cells, and the collection of vascular endothelial cells forms a reticular vascular structure.

The spheroid may contain glial cells, stem cells, and vascular endothelial cells, and the glial cells may cover at least a portion of the spheroid surface. The glial cells may be astrocytes, the vascular endothelial cells may be brain microvascular endothelial cells, and the stem cells may be adipose-derived stem cells.

A method for producing spheroids according to one aspect of the present invention comprises the step of co-culturing neurons or glial cells, stem cells, and vascular endothelial cells on a cell-adhesive cell culture substrate, wherein none of the neurons or glial cells, stem cells, and vascular endothelial cells have formed spheroids at the start of the co-culture.

The step of co-culturing neural cells or glial cells with stem cells and vascular endothelial cells may be a step of co-culturing glial cells, stem cells and vascular endothelial cells. The co-culturing may be performed in a mixed medium of a neural stem cell medium and a vascular endothelial cell medium. The co-culturing may be performed in a medium containing a Rho kinase inhibitor. The cell-adhesive cell culture substrate may be a cell culture substrate containing a fluorinated polyimide resin.

The present invention provides nervous system spheroids with a reticular vascular structure. Because nervous system spheroids with a reticular vascular structure have a structure more similar to brain tissue, they are expected to be useful in elucidating the pathological mechanisms of brain diseases and for drug screening.

FIG. 1 is a schematic cross-sectional view of a spheroid according to one embodiment of the present invention. 2A and 2B are images showing the process of spheroid formation in Example 1. FIG. 3A is a z-stack fluorescence image of the spheroid of Example 1 viewed from the side, and FIG. 3B is a horizontal cross-sectional image of the spheroid of Example 1. FIG. 4 is an image showing the process of spheroid formation in Example 2. FIG. 5A is a z-stack fluorescence image of the spheroid of Example 2 viewed from the side, and FIG. 5B is a horizontal cross-sectional image of the spheroid of Example 2. FIG. 6A is a z-stack fluorescence image of the spheroid of Example 2 viewed from the side, and FIG. 6B is a horizontal cross-sectional image of the spheroid of Example 2. FIG. 7 shows images showing the process of spheroid formation in Example 3. Figure 8(A) is a z-stack fluorescence image of the spheroid of Example 3 (ROCK inhibitor 20 μM) viewed from the side, and Figure 8(B) is a horizontal cross-sectional image of the spheroid of Example 3. FIG. 9 shows images showing the process of spheroid formation in Example 4. Figure 10(A) is a z-stack fluorescence image of the spheroid of Example 4 (ROCK inhibitor 10 μM) viewed from the side, and Figure 10(B) is a horizontal cross-sectional image of the spheroid of Example 4. Figure 11(A) is a z-stack fluorescence image of the spheroid of Example 4 (ROCK inhibitor 20 μM) viewed from the side, and Figure 11(B) is a horizontal cross-sectional image of the spheroid of Example 4.

A spheroid according to one aspect of the present invention comprises nerve cells or glial cells, stem cells, and vascular endothelial cells, which aggregate to form a reticular vascular structure.

The origin of the nerve cells is not particularly limited, and nerve cells derived from humans or non-human animals can be used. Furthermore, the nerve cells may be either central nervous system nerve cells or peripheral nervous system nerve cells, but are preferably central nervous system (brain or spinal cord-derived) nerve cells, and more preferably brain-derived nerve cells.

The glial cells may be one or more types of cells selected from the group consisting of astrocytes, microglia, and oligodendrocytes. The origin of the glial cells is not particularly limited, and glial cells derived from humans or non-human animals can be used. The glial cells may be astrocytes, such as human astrocytes.

Spheroids can contain either neurons or glial cells, or both. It is preferable that spheroids contain at least glial cells.

Stem cells may be, for example, pluripotent stem cells such as embryonic stem cells (ES cells) or iPS cells; tissue stem cells (somatic stem cells) such as neural stem cells, mesenchymal stem cells, or hematopoietic stem cells; or cancer stem cells. Mesenchymal stem cells are not particularly limited in origin, and examples include adipose-derived stem cells, bone marrow-derived stem cells, umbilical cord-derived stem cells, and dental pulp-derived stem cells. The origin of the stem cells is not particularly limited, and stem cells derived from humans or non-human animals can be used. Stem cells may be, for example, human adipose-derived stem cells (AdSCs).

The vascular endothelial cells may be brain microvascular endothelial cells, vein-derived endothelial cells, or artery-derived endothelial cells, and are preferably brain microvascular endothelial cells. The origin of the vascular endothelial cells is not particularly limited, and vascular endothelial cells derived from humans or animals other than humans can be used. The vascular endothelial cells may be, for example, human brain microvascular endothelial cells.

The spheroids may further contain cells that make up blood vessels other than vascular endothelial cells, such as pericytes and smooth muscle cells. The origin of the cells that make up blood vessels is not particularly limited, and cells derived from humans or non-human animals can be used.

The ratio of stem cells to vascular endothelial cells may be, for example, 1:1 to 10:1, and more specifically, for example, 5:1 to 10:1, 2:1 to 3:1, or 1:1 to 2:1. The ratio of vascular endothelial cells to neural cells may be, for example, 1:0.5 to 1:20, and more specifically, for example, 1:10 to 1:20, 1:5 to 1:10, or 1:0.5 to 1:3. The ratio of vascular endothelial cells to glial cells may be, for example, 1:0.5 to 1:30, and more specifically, for example, 1:20 to 1:30, 1:10 to 1:15, or 1:5 to 1:10.

The spheroids have a reticular vascular structure therein, containing the above-mentioned blood vessels and endothelial cells. As used herein, "vascular structure" encompasses both immature and mature vascular structures, and specifically refers to a structure having at least a continuous monolayer of vascular endothelial cells forming a lumen. The vascular endothelial cell layer may be covered with a basement membrane, and pericytes or smooth muscle cells may be attached thereto. A reticular vascular structure refers to a reticular structure formed by blood vessels branching or connecting to each other. While the vascular structure is preferably formed throughout the spheroid, it may also be formed locally in a portion of the spheroid. The presence of a reticular vascular structure facilitates the distribution of nutrients and oxygen throughout the spheroid. Note that spheroids having a shape including a dome portion and a flat portion, in which multiple vascular structures are formed from the flat portion along a direction perpendicular to the flat portion, are not intended to be included within the scope of this invention.

When spheroids contain glial cells, the glial cells are present so as to cover at least a portion of the spheroid surface. When the surface of a spheroid is partially or entirely covered by glial cells, proteins such as humoral factors produced by the glial cells diffuse into the interior of the spheroid, strengthening the interactions between the cells and improving the functional expression of each cell. Some glial cells may be present inside the spheroid along with stem cells.

The spheroids may be, for example, approximately spherical. However, if the spheroids are adhered to a cell culture substrate, the spheroids may have a flat portion on the adhesive surface. The size of the spheroids is not particularly limited, and the spheroids may have a diameter of, for example, 10 to 1500 μm, 10 to 1000 μm, or 10 to 800 μm. Here, the diameter of the spheroids is measured by standard methods, for example, using image analysis software or a particle size distribution analyzer.

Figure 1 shows a schematic cross-sectional view of a roughly spherical spheroid according to one embodiment. Spheroid 10 is a spheroid containing glial cells, stem cells, and vascular endothelial cells, and includes stem cell aggregate 1, a reticular vascular structure 3 formed within stem cell aggregate 1, and a layer 5 of glial cells covering the surface of stem cell aggregate 1.

The glial cell layer 5 is a continuous or discontinuous layer that covers at least a portion or the entire surface of the stem cell aggregate 1. The thickness of the glial cell layer 5 is not particularly limited, and the glial cell layer 5 may be a monolayer or a multilayer of cells. Some of the glial cells may be present within the stem cell aggregate 1, and the glial cell layer 5 may extend toward the interior of the spheroid 10.

When spheroid 10 contains neurons, the neurons form aggregate 1 with stem cells, regardless of the presence or absence of glial cells. Although spheroid 10 contains glial cells, spheroid 10 may not contain glial cells, in which case glial cell layer 5 is not formed.

In one embodiment, the spheroids may be adhered to a cell-adhesive cell culture substrate. Spheroids adhered to a cell culture substrate have the advantage that they can be safely transported without being damaged, and also have the advantage that they can be used for screening or various evaluations without being isolated.

The cell culture substrate is not particularly limited as long as it is a substrate to which cells can adhere, and may be a known substrate for adhesive culture, or more specifically, a known culture vessel for adhesive culture. In other words, part or all of the surface of the substrate has cell adhesive properties. A cell-adhesive surface is a surface to which cells can adhere at certain adhesion points when they settle on the surface in a culture medium.

More specifically, the substrate may contain a cell adhesive substance. The cell adhesive substance may be, for example, a protein or a synthetic resin. The protein may be, for example, collagen, fibronectin, or laminin. The synthetic resin may be, for example, a fluororesin, a polyimide resin, polysulfone, polyethersulfone, polydimethylsiloxane, or a mixture thereof. The synthetic resin is preferably a fluorinated polyimide resin. A fluorinated polyimide resin is, in other words, a fluorine-containing polyimide resin.

Fluorinated polyimide resins include, for example, 4,4'-hexafluoroisopropylidenediphthalic anhydride (6FDA)/1,4-bis(aminophenoxy)benzene (TPEQ) copolymer, 6FDA/1,3-bis(4-aminophenoxy)benzene (TPER) copolymer, 6FDA/4,4'-oxydiphthalic anhydride (ODPA)/TPEQ copolymer, 4,4'-(4,4'-isopropylidenediphenoxy)diphthalic acid (BPADA)/2,2-bis[4-(4-aminophenoxy)phenyl]hexafluoro The copolymer may be 6FDA/2,2-bis(4-(4-aminophenoxy)phenyl)propane (HFBAPP) copolymer, 6FDA/2,2'-bis(trifluoromethyl)benzidine (TFMB) copolymer, 6FDA/4,4'-diaminodiphenyl ether (ODA) copolymer, 6FDA/4,4'-bis(4-aminophenoxy)biphenyl (BAPB) copolymer, or 6FDA/2,2-bis(4-(4-aminophenoxy)phenyl)sulfone (BAPS) copolymer.

The weight-average molecular weight of the fluorinated polyimide resin is, for example, 5,000 to 2,000,000, preferably 8,000 to 1,000,000, and more preferably 20,000 to 500,000. In this specification, the weight-average molecular weight is measured by the following method.

(Measurement of weight average molecular weight)
Apparatus: HCL-8220GPC (manufactured by Tosoh Corporation)
Column: TSKgel Super AWM-H
Eluent (LiBr·H ₂ O, N-methylpyrrolidone containing phosphoric acid): 0.01 mol/L
Measurement method: A 0.5% by mass solution is prepared in an eluent, and the molecular weight is calculated based on a calibration curve prepared with polystyrene.

In one embodiment, the substrate may have a plurality of recesses each having an opening. The bottom surface of the recess may have cell adhesive properties, and more specifically, the bottom surface of the recess may be made of the cell adhesive substance.

The number of recesses is not particularly limited, and the number of recesses per 1 ^cm2 of the substrate may be 1 or more, 10 or more, 20 or more, 30 or more, or 50 or more, and may be 1,000 or less, 500 or less, 300 or less, 200 or less, or 100 or less. The total number of recesses in the substrate according to this embodiment may be, for example, 1 or more, 10 or more, 100 or more, 1,000 or more, 10,000 or more, or 50,000 or more.

The shape of the opening of the recess is not particularly limited and may be, for example, circular, polygonal, or elliptical. The diameter of the opening may be, for example, 2000 μm or less, 10 to 2000 μm, 10 to 1000 μm, 10 to 700 μm, 10 to 600 μm, or 10 to 500 μm. In this specification, the diameter of a certain portion refers to the diameter of a circle circumscribing that portion, i.e., the maximum length of that portion.

The shape of the bottom of the recess is not particularly limited and may be, for example, a circle, a polygon, or an ellipse. The shape of the bottom may be the same as or different from the shape of the opening. The diameter of the bottom may be the same as or different from the diameter of the opening, and may be smaller or larger than the diameter of the opening. The diameter of the bottom may be, for example, 10 to 2000 μm, 10 to 1000 μm, 10 to 700 μm, 10 to 600 μm, 10 to 500 μm, 10 to 400 μm, or 10 to 300 μm. The bottom of the recess may be a flat surface, or a flat and smooth surface.

The distance between an opening and an adjacent opening, i.e., the length of the gap, is not particularly limited. The length of the gap may be, for example, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, 300 μm or less, 200 μm or less, or 100 μm or less.

The depth of the recesses is not particularly limited and may be, for example, 100 nm to 500 nm, 10 μm to 1000 μm, or 10 μm to 300 μm.

In this embodiment, all surfaces of the substrate other than the bottom surfaces of the recesses, i.e., the inner surfaces of the recesses and the top surface of the substrate (which can also be considered the periphery of the recesses), may be non-cell-adhesive. A non-cell-adhesive surface is a surface to which cells do not adhere at all, or to which cells temporarily adhere weakly but then naturally detach. More specifically, all surfaces of the substrate other than the bottom surfaces of the recesses may be made of a non-cell-adhesive material.

The non-cell adhesive substance is not particularly limited as long as it does not bind to molecules such as proteins and sugar chains present on the surface of cells. Examples of non-cell adhesive substances include polyethylene glycol and its derivatives, 2-methacryloyloxyethyl phosphorylcholine (MPC) and its derivatives, compounds containing polyhydroxyethyl methacrylate (poly-HEMA) and its derivatives, or polymers of these compounds, compounds containing segmented polyurethane (SPC) and its derivatives, proteins such as albumin, and sugar chains to which cells do not adhere (agarose, cellulose, etc.). These substances can be used singly or in combination. Among these, MPC and its derivatives or polymers thereof are preferred, with MPC polymers being more preferred, from the standpoints of adhesion to cell adhesive sites, simplification of the substrate manufacturing process, and improved uniformity of cells obtained by culture.

In another embodiment, the substrate may be a cell culture sheet having a micropattern formed by a cell-adhesive surface and a cell-non-adhesive surface. As an example, the cell culture sheet may have a micropattern formed by a layer of cell-adhesive material and a mask of cell-non-adhesive material provided on the surface of the layer. The micropattern may be, for example, recesses. In this case, the depth of the recesses depends on the thickness of the mask of cell-non-adhesive material. Therefore, the lower limit of the depth of the recesses is the minimum thickness at which cells can recognize the non-adhesive nature of the mask. The depth of the recesses may be, for example, less than 10 μm, or may be 100 nm to 500 nm. The number of recesses, the shape and diameter of the openings and bottoms of the recesses, and the distance between the recesses may be the same as in the above embodiment.

The formation of a micropattern may be carried out by, for example, microcontact printing, spin coating, casting, roll coating, die coating, gravure coating, spray coating, bar coating, flexographic printing, dip coating, inkjet printing, or patterning. Patterning is a method of forming a micropattern by injecting a desired substance into the gaps in an uneven structure formed on the surface of a base material, utilizing the capillary force generated in the gaps.

From the viewpoint of observing the spheroids using a microscope, the thickness of the substrate is preferably 400 μm or less, more preferably 200 μm or less. The area of the substrate is not particularly limited and may be, for example, 0.01 to 10,000 ^cm2 or 0.03 to 5,000 ^cm2 .

A method for producing spheroids according to one aspect of the present invention comprises the step of co-culturing neurons or glial cells with stem cells and vascular endothelial cells on a cell-adhesive cell culture substrate. When producing spheroids containing both neurons and glial cells, the co-culturing step involves co-culturing neurons, glial cells, stem cells and vascular endothelial cells. This method makes it possible to produce spheroids according to the above aspect of the present invention. Details of the cell-adhesive cell culture substrate and each cell type are as described above.

Neurons or glial cells, stem cells, and vascular endothelial cells do not form spheroids at the start of co-culture. In other words, the co-culture process described above is not a process of fusing spheroids composed of different cells by co-culturing them.

The medium used for co-culture may be any medium containing components necessary for cell growth, such as Eagle's minimum essential medium (EMEM), Dulbecco's modified Eagle's medium (DMEM), α-MEM, Glasgow MEM (GMEM), IMDM, RPMI 1640, Ham's F-12, MCDB medium, or Williams' medium E, supplemented with components such as serum, growth factors, differentiation-inducing factors, antibiotics, hormones, amino acids, sugars, and salts as needed. From the perspective of forming a reticular vascular structure inside the spheroids, the medium is preferably a medium containing a vascular endothelial cell medium, more preferably a mixed medium of a neural stem cell medium or astrocyte medium and a vascular endothelial cell medium, and even more preferably a mixed medium of a neural stem cell medium and a vascular endothelial cell medium.

Vascular endothelial cell medium is a medium with a composition suitable for culturing vascular endothelial cells, and typically contains components such as vascular endothelial growth factor (VEGF), human epidermal growth factor (hEGF), human basic fibroblast growth factor (hFGF-β), and heparin in a basal medium. Examples of vascular endothelial cell medium that can be used include commercially available products such as EGM®-2 medium prepared using Lonza's EGM®-2 Bullet Kit® (catalog number: CC-3162). EGM®-2 medium contains hEGF, VEGF, R3 insulin-like growth factor 1 (R3 IGF-1), ascorbic acid, hydrocortisone, hFGF-β, heparin, fetal bovine serum (FBS), and antibiotics such as gentamicin.

Neural stem cell medium is a medium with a composition suitable for culturing neural stem cells, and typically contains components such as hEGF, hFGF-β, and neural survival factor-1 (NSF-1) in a basal medium. Examples of neural stem cell medium that can be used include commercially available products such as KBM Neural Stem Cell manufactured by Kohjin Bio Co., Ltd. KBM XB2 manufactured by Kohjin Bio Co., Ltd. may also be added to KBM Neural Stem Cell.

Astrocyte medium is a medium with a composition suitable for culturing astrocytes, and typically contains components such as hEGF, L-glutamic acid, and insulin in a basal medium. Examples of astrocyte medium that can be used include commercially available products such as AGM® Astrocyte Growth Medium, prepared using Lonza's AGM® BulletKit® (catalog number: CC-3186), and Sciencell's Astrocyte Medium (catalog number: 1801). AGM® Astrocyte Growth Medium contains FBS, L-glutamic acid, insulin, hEGF, ascorbic acid, and antibiotics such as gentamicin.

From the viewpoint of maintaining cell viability and cell proliferation, the medium used for co-culture preferably contains serum. Furthermore, from the viewpoint of improving the rate of spheroid formation, the medium used for co-culture preferably further contains a Rho kinase (ROCK) inhibitor. The concentration of the ROCK inhibitor in the medium may be, for example, 0.01 to 50 μM, specifically, for example, 0.01 to 1 μM or 5 to 50 μM.

The culture temperature is not particularly limited, but is usually around 25 to 40°C. The relative humidity during culture is not particularly limited, and may be, for example, 40 to 95% RH.

The culture time is not particularly limited and can be determined appropriately depending on the cell proliferation rate and the desired size of the spheroids. The culture time may be, for example, 4 hours to 30 days (4 hours to 720 hours), 1 day to 14 days (24 hours to 336 hours), or 1 day to 7 days (24 hours to 168 hours).

The ratio of stem cells to vascular endothelial cells may be, for example, 1:1 to 10:1, specifically, for example, 5:1 to 10:1, 2:1 to 3:1, or 1:1 to 2:1. The ratio of vascular endothelial cells to neural cells may be, for example, 1:0.5 to 1:20, specifically, for example, 1:10 to 1:20, 1:5 to 1:10, or 1:0.5 to 1:3. The ratio of vascular endothelial cells to glial cells may be, for example, 1:0.5 to 1:30, specifically, for example, 1:20 to 1:30, 1:10 to 1:15, or 1:5 to 1:10.

It is preferable to degas the cell culture substrate before culturing cells. The degassing method is not particularly limited, and common methods such as spraying, pipetting, shaking, heating and cooling, centrifugation, vacuum degassing, and ultrasonic treatment can be used.

<Preparation of human neuronal cells (NHN)>
^Two mL of AlphaBioCoat (catalog number: AC001, manufactured by Neuromics) was added to a culture flask (culture area: 25 cm ) and placed in a 5% (v/v) _CO incubator at 37°C for 30 minutes. The culture flask was then washed twice with 10 mL of phosphate-buffered saline (PBS), and the culture surface inside the flask was coated with AlphaBioCoat. Frozen human neurons (brain-derived; catalog number: NHC001, purchased from Neuromics) were thawed in a 37°C water bath and added to 10 mL of neuron-specific medium (catalog number: HNM001, manufactured by Neuromics). This cell suspension was added to the culture flask and cultured in a 5% (v/v) _CO incubator at 37°C. Medium changes were performed every 2-3 days. After incubation, the medium was removed from the culture flask, and 2 mL of cell detachment solution Accutase® (Promocell) was added. The culture flask was then placed in a 5% (v/v) _CO2 incubator at 37°C for approximately 5 minutes to detach the cells. The cell detachment solution was then collected in a tube, and the culture flask was washed with 8 mL of medium, which was then transferred to the tube. After centrifugation at 500 × g for 3 minutes, the cells were suspended in 0.5 mL of neural stem cell medium, and the cell number was counted.

<Preparation of human astrocytes (NHA)>
Frozen human astrocytes (catalog number: CC-2565, purchased from Lonza) were thawed in a 37°C thermostatic water bath and added to 15 mL of astrocyte-specific medium (catalog number: CC-3186, manufactured by Lonza, or catalog number: 1801, manufactured by ScienCell). This cell suspension was added to a culture flask (culture area: 75 cm ² ) and cultured in a 5% (v/v) CO ₂ incubator at 37°C. Medium changes were performed every two days. After culture, the medium was removed from the culture flask, 5 mL of cell detachment solution Accutase (registered trademark) was added, and the culture flask was left to stand in a 5% (v/v) CO ₂ incubator at 37°C for approximately 5 minutes to detach the cells. The cell detachment solution was then collected in a tube, the culture flask was washed with 10 mL of medium, and the medium was transferred to the tube. After centrifugation at 210×g for 5 minutes, the cells were suspended in 1 mL of a medium specifically for neural stem cells or astrocytes, and the number of cells was counted.

<Preparation of human adipose-derived stem cells (AdSCs)>
Frozen human adipose-derived stem cells (catalog number: PT-5006, purchased from Lonza) were thawed in a 37°C water bath and added to 9 mL of adipose-derived stem cell-specific medium (trade name: KBM ADSC-1, manufactured by Kohjin Bio Co., Ltd.) containing 5% FBS and 1% antibiotics. The cells were then centrifuged at 210 x g for 5 minutes, the supernatant removed, and the cells dispersed in 1 mL of adipose-derived stem cell-specific medium. 15 mL of this cell suspension (3.0 x ¹⁰ cells) was added to a culture flask (culture area 75 cm ² ) and cultured in a 5% (v/v) CO ₂ incubator at 37°C. The medium was changed every two days. After culturing, the medium was removed from the culture flask, and 5 mL of cell detachment solution Accutase® was added. The culture flask was left at room temperature for approximately 5 minutes to detach the cells. The cell detachment solution was then collected in a tube, the culture flask was washed with 10 mL of medium, and the medium was transferred to the tube. After centrifugation at 210×g for 5 minutes, the cells were suspended in 1 mL of astrocyte-specific medium or neural stem cell-specific medium containing KBM XB2 (Kohjin Bio Co., Ltd.), and the number of cells was counted.

<Preparation of human brain capillary endothelial cells (HBECs)>
Five mL of Attachment Factor (registered trademark) (product number: 4Z0-210, Cell Systems) was added to a culture flask (culture area 75 cm ² ) and immediately aspirated to coat the culture surface inside the flask with Attachment Factor. Frozen human brain capillary endothelial cells (product number: ACBRI 376, purchased from Cell Systems) were thawed in a 37°C thermostatic water bath and added to 15 mL of vascular endothelial cell-specific medium (product number: 4Z0-500-R, Cell Systems). This cell suspension was added to the culture flask and cultured in a 5% (v/v) CO ₂ incubator at 37°C. The medium was changed every two days. After incubation, the medium was removed from the culture flask, and 5 mL of cell detachment solution Accutase® was added. The culture flask was placed in a 37°C, 5% (v/v) _CO2 incubator for approximately 2 minutes to detach the cells. The cell detachment solution was then collected in a tube, and the culture flask was washed with 10 mL of medium, which was then transferred to the tube. After centrifugation at 210 × g for 5 minutes, the cells were suspended in 0.5 mL of astrocyte-specific medium or neural stem cell-specific medium containing KBM XB2, and the cell number was counted.

<Preparing the culture vessel>
In the following examples, a culture vessel equipped with 400 holes (cavities) with circular openings and bottoms each approximately 300 μm in diameter was used. This culture vessel contained a 6FDA/TPEQ copolymer on the bottom, and the rest of the vessel was coated with MPC polymer. The culture vessel was degassed before use. Specifically, approximately 1 mL of PBS was added to the culture vessel and pipetted. The culture vessel was then placed in a 5% (v/v) _CO2 incubator at 37°C for 15 to 30 minutes. After pipetting again, the PBS was aspirated to complete the degassing process.

<Observation of spheroids>
In the following examples, the resulting spheroids were observed as follows. First, the spheroids were fixed by treatment with 4% paraformaldehyde phosphate buffer for 15 minutes. Next, each cell was fluorescently stained. NHNs were stained with an anti-MAP2 antibody (Abcam) labeled with Alexa Fluor® 647 dye. NHAs were stained with an anti-glial fibrillary acidic protein (GFAP) antibody (Abcam) and a secondary antibody labeled with Alexa Fluor® 488 dye (Thermo Fisher). HBECs were stained with an anti-CD31 antibody (BD Biosciences) and a secondary antibody labeled with Alexa Fluor® 594 dye (Thermo Fisher). Nuclei were stained with DAPI. The stained spheroids were added with the clearing reagent SCALEVIEW®-S4 (Fujifilm Wako Pure Chemical Industries, Ltd.) and incubated overnight at 37°C. The clearing treatment enabled observation of the entire spheroid, i.e., from the bottom to the apex. The cleared spheroids were observed using a confocal laser microscope.

Example 1
NHNs were seeded at 250 cells/well, NHAs at 750 cells/well, AdSCs at 200 cells/well, and HBECs at 100 cells/well into a culture vessel. The culture vessel was left in a safety cabinet for 15 minutes and then placed in a 5% (v/v) _CO2 incubator at 37°C for 4 hours. Next, 5 mL of a mixed medium (containing 1% serum) was added to the culture vessel, which was obtained by mixing equal amounts of neural stem cell medium (KBM Neural Stem Cell supplemented with KBM XB2, both manufactured by Kohjin Bio) and vascular endothelial cell medium (catalog number CC-3162, manufactured by Lonza). The culture vessel was then placed back in a 5% (v/v) _CO2 incubator at 37°C and cultured for 3 days to produce spheroids.

The spheroid formation process is shown in Figures 2(A) and (B). Four hours after seeding, spheroids began to form in some wells (Figure 2(A)), and by day 3, spheroids had formed in all wells (Figure 2(B)). Fluorescence images of the spheroids three days after seeding are shown in Figures 3(A) and 3(B). Figure 3(A) is a z-stack of fluorescence images of HBECs viewed from the side. Figure 3(B) shows a horizontal cross-sectional image of the spheroid, showing the fluorescence (white) of the cell nucleus (top left), NHA (top center), HBEC (top right), and NHN (bottom left), as well as a bright-field image of the spheroid (bottom center). As these images show, a network of vascular structures with lumens formed by HBECs had formed within the spheroids. NHA was also present on the surface and inside the spheroids.

The particle size of the spheroids was measured from microscopic images using image analysis software (WinROOF, manufactured by Mitani Shoji Co., Ltd.). The particle size of the spheroids was 206 ± 29 μm (n = 100).

Example 2
Spheroids were prepared in the same manner as in Example 1, except that the cells to be seeded were changed to NHA at 1,000 cells/well, AdSC at 200 cells/well, and HBEC at 100 or 200 cells/well, and the volume of the mixed medium was changed to 4 mL.

The spheroid formation process is shown in Figure 4. Four hours after seeding, spheroids began to form in some wells, and by day 3, spheroids had formed in all wells. Fluorescent images of spheroids 3 days after seeding when 100 HBECs were seeded per well are shown in Figures 5(A) and (B), and fluorescent images of spheroids 3 days after seeding when 200 HBECs were seeded per well are shown in Figures 6(A) and (B). Figures 5(A) and 6(A) are lateral z-stack images of HBEC fluorescence images. Figures 5(B) and 6(B) show horizontal cross-sectional images of the spheroid, showing the fluorescence (white) of the cell nucleus (upper left), NHA (upper right), and HBEC (lower left), as well as a bright-field image of the spheroid (lower right). As these images show, regardless of the number of HBECs seeded, a network of vascular structures with lumens formed by HBECs was formed within the spheroids. Furthermore, NHA was present, covering the surface of the spheroids.

Example 3
Spheroids were prepared in the same manner as in Example 2, except that a ROCK inhibitor (Y-27632, Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the mixed medium at a final concentration of 10 or 20 μM. The seeding number of HBECs was adjusted to 100 cells/well.

The process of spheroid formation is shown in Figure 7. Four hours after seeding, spheroids began to form in some wells, and by day 3, spheroids had formed in all wells. Fluorescence images of spheroids (medium containing 20 μM of ROCK inhibitor) three days after seeding are shown in Figure 8 (A) and (B). Figure 8 (A) is a z-stack of fluorescence images of HBECs viewed from the side. Figure 8 (B) shows a horizontal cross-sectional image of the spheroid, showing the fluorescence (white) of the cell nuclei (top left), NHA (top right), and HBECs (bottom left), as well as a bright-field image of the spheroid (bottom right). As these images show, a reticular vascular structure with lumens formed by HBECs was formed within the spheroid. NHA was present, covering the surface of the spheroid. Some NHA was also present inside the spheroid.

The spheroid formation rate was calculated according to the following formula.
Spheroid formation rate (%) = {number of spheroids (pieces)} / {total number of holes (pieces)} × 100
The spheroid formation rate was 99% when the ROCK inhibitor concentration was 10 μM and 84% when it was 20 μM. For comparison, when a similar experiment was performed without adding a ROCK inhibitor to the medium, the spheroid formation rate was 74%. Although a ROCK inhibitor was present at 1% in the mixed medium of neural stem cell medium and vascular endothelial cell medium, it was found that adding the ROCK inhibitor to the medium suppressed excessive cell movement and improved the spheroid formation rate.

Example 4
Spheroids were prepared in the same manner as in Example 1, except that a ROCK inhibitor (Y-27632, Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the mixed medium at a final concentration of 10 or 20 μM, and the spheroid formation rate was calculated in the same manner as in Example 3. The seeding number of HBECs was adjusted to 100 cells/well.

The spheroid formation process is shown in Figure 9. Four hours after seeding, spheroids began to form in some wells, and by day 3, spheroids had formed in all wells. Spheroids formed under all conditions, regardless of the ROCK inhibitor concentration. Fluorescence images of spheroids three days after seeding are shown in Figures 10 (ROCK inhibitor 10 μM) and 11 (ROCK inhibitor 20 μM) (A) and (B). Figures 10 and 11 (A) are lateral z-stack images of HBEC fluorescence images. Figures 10 and 11 (B) show horizontal cross-sectional images of the spheroid, showing the fluorescence (white) of the cell nucleus (top left), NHA (top center), HBEC (top right), and NHN (bottom left), as well as a bright-field image of the spheroid (bottom center). As these images show, a network of vascular structures with lumens formed by HBECs was formed within the spheroid. Furthermore, NHA was present, covering the surface of some of the spheroids. Some NHA was also present inside the spheroids. NHN was present near the surface and inside the spheroids. The spheroid formation rate was 95% when the ROCK inhibitor concentration was 10 μM and 92% when it was 20 μM. For comparison, when a similar experiment was performed without adding a ROCK inhibitor to the medium, the spheroid formation rate was 75%. It was found that when nerve cells were used in combination with the cells of Example 3, adding a ROCK inhibitor to the medium suppressed excessive cell movement and improved the spheroid formation rate.

1...Stem cell aggregate, 3...Reticulated vascular structure, 5...Glial cell layer, 10...Spheroid.

Claims (10)

A spheroid comprising nerve cells or glial cells, stem cells, and vascular endothelial cells, wherein the collection of vascular endothelial cells forms a reticular vascular structure. glial cells, stem cells, and vascular endothelial cells,
The spheroid of claim 1 , wherein the glial cells cover at least a portion of the surface of the spheroid. The spheroid of claim 2, wherein the glial cells are astrocytes. The spheroid according to any one of claims 1 to 3, wherein the vascular endothelial cells are brain microvascular endothelial cells. The spheroid according to any one of claims 1 to 4, wherein the stem cells are adipose-derived stem cells. The method comprises a step of co-culturing nerve cells or glial cells, stem cells, and vascular endothelial cells on a cell-adhesive cell culture substrate,
The method for producing spheroids according to any one of claims 1 to 5, wherein each of the nerve cells or glial cells, the stem cells, and the vascular endothelial cells does not form a spheroid at the start of co-culture. The method according to claim 6, wherein the step of co-culturing nerve cells or glial cells with stem cells and vascular endothelial cells is a step of co-culturing glial cells with stem cells and vascular endothelial cells. The method according to claim 6 or 7, wherein the co-culture is carried out in a mixed medium of a medium for neural stem cells and a medium for vascular endothelial cells. The method according to any one of claims 6 to 8, wherein the co-culture is carried out in a medium containing a Rho kinase inhibitor. The method according to any one of claims 6 to 9, wherein the cell-adhesive cell culture substrate is a cell culture substrate containing a fluorinated polyimide resin.