Spheroids and methods for producing same

Abstract

To provide a nervous system spheroid comprising a reticular vessel structure inside.SOLUTION: A spheroid according to one aspect of the present invention comprises neurons or glial cells, stem cells, and vascular endothelial cells, with a population of the vascular endothelial cells forming a reticular vessel structure.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to spheroids and methods for producing the same. [Background technology]

[0002] In recent years, attention has been focused on the creation of tissues mimicking the structure of organs by three-dimensional cell culture. For example, nervous system spheroids containing nervous system cells and vascular 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 composed of neural progenitor cells derived from induced pluripotent stem cells (iPS cells) with spheroids composed of vascular endothelial cells. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] L. Song et al., Sci. Rep. 9, 5977(2019) Summary of the Invention [Problem to be solved by the invention]

[0004] In conventional nervous system spheroids containing nervous system cells and vascular cells, the vascular structure is not formed in a reticular pattern. The present invention has been made in view of this problem, and aims to provide a nervous system spheroid having a reticular vascular structure. [Means for solving the problem]

[0005] A spheroid according to one aspect of the present invention comprises nerve cells or glial cells, stem cells, and vascular endothelial cells, and the aggregation of the vascular endothelial cells forms a reticular vascular structure.

[0006] The spheroid may comprise glial cells, stem cells, and vascular endothelial cells, and the glial cells may cover at least a portion of the surface of the spheroid. 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.

[0007] A method for producing spheroids according to one aspect of the present invention comprises a 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.

[0008] 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 carried out in a mixed medium of a neural stem cell medium and a vascular endothelial cell medium. The co-culturing may be carried out 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. [Effects of the Invention]

[0009] The present invention provides a neural spheroid having a reticular vascular structure. The neural spheroid having a reticular vascular structure is more similar to brain tissue, and is therefore expected to be useful in elucidating the pathological mechanisms of brain diseases and in drug screening. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic cross-sectional view of a spheroid according to one embodiment of the present invention. [Figure 2] 2(A) and (B) are images showing the process of spheroid formation in Example 1. [Figure 3] FIG. 3(A) is a z-stack fluorescence image of the spheroid of Example 1 viewed from the side, and FIG. 3(B) is a horizontal cross-sectional image of the spheroid of Example 1. [Figure 4] FIG. 4 shows images showing the process of spheroid formation in Example 2. [Figure 5] 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. [Figure 6] FIG. 6(A) is a z-stack fluorescence image of the spheroid of Example 2 viewed from the side, and FIG. 6(B) is a horizontal cross-sectional image of the spheroid of Example 2. [Figure 7] FIG. 7 shows images showing the process of spheroid formation in Example 3. [Figure 8] Figure 8(A) is a lateral view of a z-stack of fluorescent images of the spheroid of Example 3 (ROCK inhibitor 20 μM), and Figure 8(B) is a horizontal cross-sectional image of the spheroid of Example 3. [Figure 9] FIG. 9 shows images showing the process of spheroid formation in Example 4. [Figure 10] Figure 10(A) is a lateral view of a z-stack of fluorescent images of the spheroid of Example 4 (ROCK inhibitor 10 μM), and Figure 10(B) is a horizontal cross-sectional image of the spheroid of Example 4. [Figure 11] Figure 11 (A) is a lateral view of a z-stack of fluorescent images of the spheroid of Example 4 (ROCK inhibitor 20 μM), and Figure 11 (B) is a horizontal cross-sectional image of the spheroid of Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0011] A spheroid according to one aspect of the present invention comprises nerve cells or glial cells, stem cells, and vascular endothelial cells, and the vascular endothelial cells aggregate to form a reticular vascular structure.

[0012] The origin of the nerve cells is not particularly limited, and nerve cells derived from humans or animals other than humans can be used. 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.

[0013] 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.

[0014] The spheroids may contain either nerve cells or glial cells, or both, and preferably contain at least glial cells.

[0015] 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 may be cancer stem cells. The origin of mesenchymal stem cells is not particularly limited, 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).

[0016] 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.

[0017] The spheroids may further contain cells that constitute blood vessels other than vascular endothelial cells, such as pericytes, smooth muscle cells, etc. The origin of the cells that constitute blood vessels is not particularly limited, and cells derived from humans or animals other than humans can be used.

[0018] 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 nerve 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.

[0019] The spheroid has a reticular vascular structure therein, including the above-mentioned blood vessels and endothelial cells. As used herein, the term "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. The reticular vascular structure refers to a reticular structure formed by blood vessels branching or connecting to each other. The vascular structure is preferably formed throughout the spheroid, but may also be formed locally in a portion of the spheroid. Having 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 the present invention.

[0020] When the spheroid contains glial cells, the glial cells are present so as to cover at least a portion of the spheroid surface. When the surface of the spheroid is partially or entirely covered with glial cells, proteins such as humoral factors produced by the glial cells diffuse into the interior of the spheroid, strengthening the interaction between the cells and improving the functional expression of each cell. Some glial cells may be present inside the spheroid together with stem cells.

[0021] The spheroids may be, for example, approximately spherical. However, when the spheroids are adhered to a cell culture substrate, the spheroids may have a flat portion on the adhesion 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 a conventional method, for example, using image analysis software or a particle size distribution analyzer.

[0022] 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.

[0023] 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.

[0024] When spheroid 10 contains neurons, the neurons form aggregates 1 together 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.

[0025] In one embodiment, the spheroids may be adhered to a cell-adhesive cell culture substrate, which has the advantage that the spheroids can be safely transported without being damaged, and also has the advantage that the spheroids can be used for screening or various evaluations without being isolated.

[0026] 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, more specifically, a known culture vessel for adhesive culture. That is, 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.

[0027] 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. In other words, a fluorinated polyimide resin is a fluorine-containing polyimide resin.

[0028] Examples of the fluorinated polyimide resin include 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]hexafluoroisopropylidenediphthalic anhydride (6FDA ... The copolymer may be a 6FDA / 2,2-bis(4-(4-aminophenoxy)phenyl)propane (HFBAPP) copolymer, a 6FDA / 2,2'-bis(trifluoromethyl)benzidine (TFMB) copolymer, a 6FDA / 4,4'-diaminodiphenyl ether (ODA) copolymer, a 6FDA / 4,4'-bis(4-aminophenoxy)biphenyl (BAPB) copolymer, or a 6FDA / 2,2-bis(4-(4-aminophenoxy)phenyl)sulfone (BAPS) copolymer.

[0029] 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.

[0030] (Measurement of weight average molecular weight) Device: HCL-8220GPC (Tosoh Corporation) Column: TSKgel Super AWM-H Eluent (LiBr·H2O, N-methylpyrrolidone with phosphoric acid): 0.01 mol / L Measurement method: A 0.5% by mass solution is prepared using an eluent, and the molecular weight is calculated based on a calibration curve prepared using polystyrene.

[0031] In one embodiment, the substrate may include a plurality of recesses each having an opening, and the bottom surface of each recess may have cell adhesive properties, more specifically, the bottom surface of each recess may be made of the cell adhesive substance.

[0032] The number of recesses is not particularly limited, and may be within 1 cm of the substrate. 2 The number of recesses per unit area 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.

[0033] The shape of the opening of the recess is not particularly limited and may be, for example, a circle, a polygon, or an ellipse. 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 the portion, i.e., the maximum length of the portion.

[0034] 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.

[0035] The distance between adjacent openings, i.e., the length of the gap, is not particularly limited and 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.

[0036] 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.

[0037] 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 composed of a non-cell-adhesive material.

[0038] 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 improvement of cell uniformity obtained by culture.

[0039] 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. For 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, and 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.

[0040] The formation of the fine pattern 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. The patterning method is a method of forming a fine pattern by injecting a desired substance into gaps in an uneven structure formed on the surface of a base material by utilizing capillary force generated in the gaps.

[0041] 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 cm. 2 or 0.03 to 5000 cm 2 It may be.

[0042] A method for producing spheroids according to one aspect of the present invention comprises a 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 allows the production of spheroids according to the above aspect of the present invention. The details of the cell-adhesive cell culture substrate and each cell type are as described above.

[0043] Neither the nerve cells or glial cells, the stem cells, nor the vascular endothelial cells formed spheroids at the start of the co-culture, i.e., the co-culture step was not a step of fusing spheroids composed of different cells by co-culturing them.

[0044] 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 serum, growth factors, differentiation-inducing factors, antibiotics, hormones, amino acids, sugars, salts, and other components as needed. To form a reticular vascular structure within the spheroids, the medium is preferably a medium containing a vascular endothelial cell medium, more preferably a mixture of a neural stem cell medium or astrocyte medium with a vascular endothelial cell medium, and even more preferably a mixture of a neural stem cell medium and a vascular endothelial cell medium.

[0045] 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 include commercially available EGM®-2 medium prepared using Lonza's EGM®-2 BulletKit® (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.

[0046] 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). KBM Neural Stem Cell may be supplemented with KBM XB2 (manufactured by Kohjin Bio).

[0047] Astrocyte medium is a medium with a composition suitable for culturing astrocytes, typically containing components such as hEGF, L-glutamic acid, and insulin in a basal medium. Commercially available astrocyte medium includes, for example, 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.

[0048] 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 spheroid formation rate, 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.

[0049] The culture temperature is not particularly limited, but is usually about 25 to 40° C. The relative humidity during culture is not particularly limited, and may be, for example, 40 to 95% RH.

[0050] 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).

[0051] 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 nerve 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.

[0052] 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. [Example]

[0053] <Preparation of human neuronal cells (NHN)> Culture flask (culture area 25 cm 2Two mL of AlphaBioCoat (catalog number: AC001, Neuromics) was added to the flask and incubated in a 5% (v / v) CO2 incubator at 37°C for 30 minutes. The flask was then washed twice with 10 mL of phosphate-buffered saline (PBS), and the culture surface of the flask was coated with AlphaBioCoat. Frozen human neuronal cells (brain-derived; catalog number: NHC001, purchased from Neuromics) were thawed in a 37°C water bath and added to 10 mL of neuronal culture medium (catalog number: HNM001, Neuromics). This cell suspension was added to the culture flask and cultured in a 5% (v / v) CO2 incubator at 37°C. The medium was changed every 2–3 days. After culturing, the medium was removed from the culture flask, and 2 mL of cell detachment solution Accutase (registered trademark) (Promocell) was added. The culture flask was 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-specific medium, and the cell number was counted.

[0054] <Preparation of human astrocytes (NHA)> Frozen human astrocytes (catalog number CC-2565, purchased from Lonza) were thawed in a 37°C water bath and added to 15 mL of astrocyte-specific medium (catalog number CC-3186, Lonza, or catalog number 1801, ScienCell). This cell suspension was placed in a culture flask (culture area 75 cm). 2 ) and cultured in a 5% (v / v) CO2 incubator at 37°C. The medium was changed every two days. After culturing, 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 standing 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, 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 neural stem cell-specific medium or astrocyte-specific medium, and the cell number was counted.

[0055] <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) containing 5% FBS and 1% antibiotics. The cells were then centrifuged at 210 × g for 5 minutes, after which the supernatant was removed and the cells were dispersed in 1 mL of adipose-derived stem cell-specific medium. 15 mL (3.0 × 10 5 Cells) were placed in a culture flask (culture area 75 cm 2 ) and cultured in a 5% (v / v) CO2 incubator at 37°C. The medium was changed every two days. After culturing, 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 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 (manufactured by Kohjin Bio Co., Ltd.), and the cell number was counted.

[0056] <Preparation of human brain capillary endothelial cells (HBEC)> Culture flask (culture area 75 cm 25 mL of Attachment Factor® (product number: 4Z0-210, Cell Systems) was added to the flask and immediately aspirated, coating the culture surface with Attachment Factor. Frozen human brain capillary endothelial cells (product number: ACBRI 376, purchased from Cell Systems) were thawed in a 37°C water bath and added to 15 mL of 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) CO2 incubator at 37°C. The medium was changed every two days. After culture, the medium was removed from the culture flask, 5 mL of cell detachment solution Accutase® was added, and the culture flask was left to stand in a 5% (v / v) CO2 incubator at 37°C for approximately 2 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 0.5 mL of astrocyte-specific medium or neural stem cell-specific medium containing KBM XB2, and the cell number was counted.

[0057] <Preparing the culture vessel> In the following examples, a culture vessel with 400 holes (cavities) with a circular opening and bottom surface of approximately 300 μm in diameter was used. The bottom surface of this culture vessel contained a 6FDA / TPEQ copolymer, and the rest of the vessel was coated with an MPC polymer. The culture vessel was degassed before use. Specifically, approximately 1 mL of PBS was added to the culture vessel and pipetted, and the culture vessel was left to stand 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.

[0058] <Observation of spheroids> In the following examples, the obtained spheroids were observed as follows. First, the spheroids were fixed by treatment with 4% paraformaldehyde phosphate buffer for 15 minutes. Next, each cell type 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 made it possible to observe the entire spheroid, from the bottom to the top. The cleared spheroid was observed using a confocal laser microscope.

[0059] Example 1 NHNs (250 cells / well), NHAs (750 cells / well), AdSCs (200 cells / well), and HBECs (100 cells / well) were seeded 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. The culture vessel was then placed in a 5% (v / v) CO2 incubator at 37°C for 3 days. The mixed medium was a mixture of equal parts neural stem cell medium (KBM Neural Stem Cell supplemented with KBM XB2, both manufactured by Kohjin Bio) and endothelial cell medium (Cat. No. CC-3162, manufactured by Lonza). The resulting mixture was then mixed with 5 mL of a 1% serum-containing medium. The culture vessel was then placed in a 5% (v / v) CO2 incubator at 37°C for 3 days to produce spheroids.

[0060] The spheroid formation process is shown in Figure 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 3 days after seeding are shown in Figure 3 (A) and (B). Figure 3 (A) shows 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 nuclei (top left), NHA (top center), HBECs (top right), and NHNs (bottom left), as well as a bright-field image of the spheroid (bottom center). As shown in these images, a network of vascular structures with lumens formed by HBECs was formed within the spheroids. NHA was also present on the surface and interior of the spheroids.

[0061] The particle size of the spheroids was measured from microscopic images using image analysis software (WinROOF, manufactured by Mitani Shoji Co., Ltd.) and was found to be 206 ± 29 μm (n = 100).

[0062] <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.

[0063] 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 Figure 5 (A) and (B), and fluorescent images of spheroids 3 days after seeding when 200 HBECs were seeded per well are shown in Figure 6 (A) and (B). Figures 5 (A) and 6 (A) are lateral z-stack images of fluorescent images of HBECs. Figures 5 (B) and 6 (B) show horizontal cross-sectional images of the spheroids, showing the fluorescence (white) of the cell nuclei (upper left), NHA (upper right), and HBECs (lower left), as well as a bright-field image of the spheroid (lower right). As shown in these images, regardless of the number of HBECs seeded, a network of vascular structures with lumens formed by HBECs was formed within the spheroids, and NHA was present covering the surface of the spheroids.

[0064] 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.

[0065] The spheroid formation process 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 (in which 20 μM of ROCK inhibitor was added to the medium) taken 3 days after seeding are shown in Figure 8 (A) and (B). Figure 8 (A) shows 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 network of vascular structures with lumens formed by HBECs had formed within the spheroids. NHA was also present covering the surface of the spheroids. Some NHA was also present inside the spheroids.

[0066] The spheroid formation rate was calculated according to the following formula. Spheroid formation rate (%) = {number of spheroids (pieces)} / {total number of holes (pieces)} x 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, adding the ROCK inhibitor to the medium suppressed excessive cell movement and improved the spheroid formation rate.

[0067] 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.

[0068] 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 3 days after seeding are shown in Figure 10 (ROCK inhibitor 10 μM) and Figure 11 (ROCK inhibitor 20 μM) (A) and (B). Figures 10 and 11 (A) show z-stack fluorescence images of HBECs viewed from the side. Figures 10 and 11 (B) show horizontal cross-sectional images of the spheroids, showing the fluorescence (white) of the cell nuclei (top left), NHA (top center), HBECs (top right), and NHNs (bottom left), as well as a bright-field image of the spheroid (bottom center). As shown in these images, a network of vascular structures with lumens formed by HBECs was formed within the spheroids. Furthermore, NHA partially covered the surface 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 also 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. [Explanation of symbols]

[0069] 1···Stem cell aggregate, 3···Reticulated vascular structure, 5···Glial cell layer, 10···Spheroid.

Claims

1. comprising nerve cells or glial cells, stem cells, and vascular endothelial cells; and A spheroid in which the aggregation of vascular endothelial cells forms a reticular vascular structure.

2. 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.

3. The spheroid of claim 2 , wherein the glial cells are astrocytes.

4. The spheroid according to any one of claims 1 to 3, wherein the vascular endothelial cells are cerebral microvascular endothelial cells.

5. The spheroid according to any one of claims 1 to 4, wherein the stem cells are adipose-derived stem cells.

6. 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.

7. The method according to claim 6, wherein the step of co-culturing nerve cells or glial cells, stem cells, and vascular endothelial cells is a step of co-culturing glial cells, stem cells, and vascular endothelial cells.

8. 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.

9. 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.

10. The method according to any one of claims 6 to 9, wherein the cell-adhesive cell culture substrate is a cell culture substrate comprising a fluorinated polyimide resin.

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