Spheroids and method for producing same

Abstract

To provide a spheroid having terminals of a vascular structure formed on a spheroid.SOLUTION: A spheroid 100 is formed by aggregation of stem cells and vascular endothelial cells. The spheroid 100 has a shape including a dome part 10 and a flat part 20. A plurality of vascular structures 30 are formed from the flat part 20 along the perpendicular direction to the flat part 20.SELECTED DRAWING: Figure 8

Description

[Technical field]

[0001] The present invention relates to a spheroid and a method for producing the same. [Background technology]

[0002] In recent years, attention has been focused on the formation of various morphologies of spheroids by three-dimensionally culturing cells. For example, a method has been proposed in which biological tissue is co-cultured with vascular cells and mesenchymal cells to biologically impart a vascular system (Patent Document 1). Biological tissues imparted with a vascular system are expected to be applied to organ transplantation, drug discovery screening, and the like. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2015 / 012158 Summary of the Invention [Problem to be solved by the invention]

[0004] In conventional spheroids with vascular structures, the vascular structures are randomly formed inside the spheroids, and the ends of the vascular structures are not formed on the surface of the spheroids. Therefore, for example, it is expected that the possibility of the spheroids being able to take root when transplanted is not necessarily high.

[0005] The present invention has been made in consideration of the above problems, and provides a spheroid in which the ends of a vascular structure are formed on the surface of the spheroid. [Means for solving the problem]

[0006] The inventors discovered that by culturing stem cells and vascular endothelial cells on a cell-adhesive cell culture substrate, it is possible to produce spheroids in which the ends of vascular structures are formed on the surface of the spheroids, and thus completed the present invention.

[0007] That is, the spheroid according to one embodiment of the present invention is an aggregate of stem cells and vascular endothelial cells, has a shape including a dome portion and a flat portion, and has a plurality of vascular structures formed from the flat portion along a direction perpendicular to the flat portion. Since the ends of the vascular structures are formed on the flat portion, which is the surface of the spheroid, such a spheroid is highly likely to be able to take root when transplanted.

[0008] A method for producing spheroids according to one embodiment of the present invention includes a step of co-culturing stem cells and vascular endothelial cells on a cell-adhesive cell culture substrate. The above-mentioned spheroids can be produced by this method. Effect of the Invention

[0009] The spheroids of the present invention have the ends of the vascular structures formed on the flat surface of the spheroid, and therefore have a high possibility of survival when transplanted. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows micrographs of spheroids produced by culturing AdSCs alone or co-culture of AdSCs and HUVECs. [Diagram 2] Figure 2 is a graph showing the average particle size of spheroids produced by culturing AdSCs alone or co-culturing AdSCs and HUVECs. *: p<0.05 (multiple comparison test by Tukey-Kramer method). [Diagram 3] FIG. 3 shows micrographs of stained spheroids produced by culturing AdSCs alone or co-culture of AdSCs and HUVECs. [Figure 4] Figure 4 shows graphs showing (A) the percentage of HUVECs and (B) the cell viability of spheroids prepared by culturing AdSCs alone or co-culture of AdSCs and HUVECs. **: p<0.01 (multiple comparison test by Tukey-Kramer method). ns: no significant difference. [Diagram 5]Figure 5 shows z-stacks of stained images of spheroids produced by co-culture of AdSCs and HUVECs (100 cells / well). (A), (B), and (C) are lateral, transverse, and top-down images of z-stacks of HUVEC staining, respectively. (D) is a bottom-down image of z-stacks of stained images of HUVECs and cell nuclei. (E) is a vertical and horizontal cross-sectional image of the spheroid. [Figure 6] Figure 6 shows horizontal cross-sectional images of spheroids produced by co-culture of AdSCs and HUVECs. In (A), HUVECs, laminin, and cell nuclei are stained, and in (B), HUVECs, type IV collagen, and cell nuclei are stained. [Figure 7] 7 is a graph showing the fluorescence intensity of spheroids produced by culturing AdSCs alone or co-culturing AdSCs and HUVECs. **: p<0.01 (multiple comparison test by Tukey-Kramer method). ns: no significant difference. [Figure 8] FIG. 8 is a schematic diagram of a spheroid. [Figure 9] FIG. 9 is a fluorescent photograph of the spheroids shown in FIG. 7, the fluorescence intensity of which was measured. [Figure 10] Figure 10 shows z-stacks of stained images of spheroids produced by co-culture of AdSCs and HUVECs (50 cells / well). (A), (B), and (C) are lateral, lateral, and top views of z-stacks of HUVEC stained images, respectively, (D) is a bottom view of z-stacks of stained images of HUVECs and cell nuclei, and (E) is a vertical and horizontal cross-sectional image of the spheroid. [Figure 11] Figure 11 shows z-stacks of stained images of spheroids produced by co-culture of AdSCs and HUVECs (200 cells / well). (A), (B), and (C) are lateral, lateral, and top views of z-stacks of HUVEC stained images, respectively, (D) is a bottom view of z-stacks of stained images of HUVECs and cell nuclei, and (E) is a vertical and horizontal cross-sectional image of the spheroid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings.

[0012] FIG. 8 is a schematic diagram of a spheroid 100 according to one embodiment of the present invention. The spheroid 100 is a cell mass in which stem cells and vascular endothelial cells are aggregated. As shown in FIG. 8, the spheroid 100 is composed of a dome portion 10 and a flat portion 20. A plurality of vascular structures 30 are formed from the flat portion 20 along a direction perpendicular to the flat portion 20. One end of the vascular structure 30 is connected to the surface of the spheroid at the flat portion 20. Therefore, when the spheroid is transplanted, there is a high possibility that the vascular structure 30 can be engrafted in the blood vessels in the living body. The vascular structure 30 is composed of vascular endothelial cells.

[0013] The size of the spheroids is not particularly limited, and may be, for example, 1 to 500 μm or 10 to 300 μm in diameter. The particle size can be measured by image analysis in accordance with the method described in Example 1.

[0014] "A direction perpendicular to the flat portion 20" refers to a direction perpendicular to the plane formed by the flat portion 20. "Along the vertical direction" means that the directional component in which the vascular structure 30 is formed includes at least a vertical component. Note that in a state in which the vascular structure 30 is formed only within the plane formed by the flat portion 20, the directional component in which the vascular structure 30 is formed includes only directional components within the plane, and therefore does not include a vertical component, and does not correspond to a state in which the vascular structure 30 is formed "along the vertical direction."

[0015] For example, the vascular structure may be formed parallel to the vertical direction, or may be formed in a state of inclination at a predetermined rising angle with respect to the vertical direction. The "rising angle" here refers to the rising portion with respect to the flat portion, i.e., the angle that the root portion of the vascular structure makes with respect to the flat portion. The rising angle is not particularly limited, but is preferably a predetermined angle from the viewpoint of supplying oxygen and nutrients to the center of the spheroid. For example, the rising angle is preferably 1 degree or more, more preferably 45 degrees or more. The maximum value of the rising angle is 90 degrees.

[0016] In addition, as long as the directional component in which the vascular structure is formed includes a vertical component, there is no particular limitation on how the vascular structure is formed, and the vascular structure does not necessarily have to be substantially linear. For example, the vascular structure may be curved (curved, spiral, etc.). The shape of the vascular structure may vary depending on the location. For example, the vascular structure may be substantially linear near the base of the vascular structure, and curved near the middle and tip (the end on the side other than the base) of the vascular structure. Furthermore, the vascular structure may include a branched structure in the middle. The branched vascular structure may be linear or curved. The branched vascular structure may be connected to other vascular structures (including branched and non-branched vascular structures).

[0017] The presence of multiple vascular structures 30 in the spheroid 100 facilitates the distribution of oxygen, nutrients, and the like throughout the spheroid 100. Preferably, the multiple vascular structures 30 are formed in a dome shape as a whole, and the dome portion is constructed by the presence of stem cells surrounding the vascular structures 30. For example, the proportion of vascular endothelial cells in the spheroid 100 may be 5% to 25% or 10% to 20%. The proportion of vascular endothelial cells in the spheroid 100 can be measured according to the method described in Example 2.

[0018] It is preferable that a basement membrane is formed on the outer periphery of the vascular structure 30. The vascular structure 30 having a basement membrane formed on the outer periphery can be regarded as a mature vascular structure, and is expected to function normally as a blood vessel. Whether or not a basement membrane is formed can be determined by confirming the presence or absence of one or more of type IV collagen, laminin, and proteoglycan.

[0019] In one embodiment, the spheroid 100 is adhered to a cell-adhesive cell culture substrate. In this case, the spheroid 100 is adhered to the cell culture substrate at the flat portion 20. The spheroid 100 adhered to the cell culture substrate has an advantage that the spheroid 100 can be safely transported without being damaged, and also has an advantage that the spheroid 100 can be used as is for drug discovery screening without being isolated.

[0020] The cell-adhesive cell culture substrate is not particularly limited as long as it is a substrate to which cells can adhere, and may be a substrate for known adhesion culture, more specifically, a culture vessel for known adhesion culture. That is, a part or the whole of the surface of the substrate has cell adhesiveness. A cell-adhesive surface is a surface to which cells can adhere at certain adhesion points when the cells settle on the surface in a culture medium.

[0021] More specifically, the substrate may include 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.

[0022] 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[ 6FDA / 2,2-bis(4-(4-aminophenoxy)phenyl)hexafluoropropane (HFBAPP), 6FDA / 2,2-bis(4-(4-aminophenoxy)phenyl)propane (BAPP) copolymer, 6FDA / 2,2'-bis(trifluoromethyl)benzidine (TFMB) copolymer, 6FDA / 4,4'-diaminodiphenyl ether (ODA) copolymer, or 6FDA / 4,4'-bis(4-aminophenoxy)biphenyl (BAPB) copolymer.

[0023] 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.

[0024] (Measurement of weight average molecular weight) Equipment: 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.

[0025] In one embodiment, the substrate may include 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.

[0026] The number of recesses is not particularly limited, and may be within 1 cm2 The number of recesses per unit area may be 10 or more, 20 or more, 30 or more, or 50 or more, and may be 1000 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, 10 or more, 100 or more, 1000 or more, 10000 or more, or 50000 or more.

[0027] 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 part refers to the diameter of a circle circumscribing the part, that is, the maximum length of the part.

[0028] 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.

[0029] 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.

[0030] The depth of the recesses is not particularly limited, and may be, for example, 100 to 500 nm, 10 to 1000 μm, or 10 to 300 μm.

[0031] In this embodiment, the entire surface of the substrate other than the bottom surface of the recess, i.e., the inner side of the recess and the upper surface of the substrate (which can also be said to be the periphery of the recess), may be cell non-adhesive. A cell non-adhesive surface is a surface to which cells do not adhere at all or to which cells naturally detach even if they temporarily adhere weakly. More specifically, the entire surface of the substrate other than the bottom surface of the recess may be composed of a cell non-adhesive material.

[0032] 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. The non-cell adhesive substance may be, for example, polyethylene glycol or a derivative thereof, 2-methacryloyloxyethyl phosphorylcholine (MPC), polyhydroxyethyl methacrylate (poly-HEMA), segmented polyurethane (SPC), or a protein such as albumin.

[0033] In another embodiment, the substrate may be a cell culture sheet having a micropattern formed of a cell adhesive surface and a cell non-adhesive surface. As an example, the cell culture sheet may have a micropattern formed of a layer of a cell adhesive material and a mask of a cell non-adhesive material provided on the surface of the layer. The micropattern may be, for example, a recess. In this case, the depth of the recess depends on the thickness of the mask of the cell non-adhesive material. Therefore, the lower limit of the depth of the recess is the minimum thickness at which cells can recognize the non-adhesiveness of the mask. The depth of the recess 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 opening and bottom of the recesses, and the distance between the recesses may be the same as in the above embodiment.

[0034] The formation of the fine pattern may be performed by, for example, a microcontact printing method, a spin coating method, a casting method, a roll coating method, a die coating method, a gravure coating method, a spray coating method, a bar coating method, a flexographic printing method, a dip coating method, an inkjet method, or a patterning method. The patterning method is a method of forming a fine pattern by injecting a desired substance into gaps in a concave-convex structure formed on the surface of a base material by utilizing capillary force generated in the gaps.

[0035] 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 0.03~5000cm 2 It may be.

[0036] The stem cells used may be derived from humans or animals other than humans, and may be, for example, ES cells, iPS cells, neural stem cells, mesenchymal stem cells, tissue stem cells (somatic stem cells), hematopoietic stem cells, or cancer stem cells, and specifically, may be, for example, human adipose derived stem cells (AdSCs).

[0037] The vascular endothelial cells used may be derived from humans or animals other than humans. Specifically, for example, the vascular endothelial cells may be human umbilical vein endothelial cells (HUVEC).

[0038] The spheroids of the present invention can be prepared by co-culturing stem cells and vascular endothelial cells on a cell-adhesive cell culture substrate. The medium used for co-culture is not particularly limited, and any cell culture basal medium, differentiation medium, or medium dedicated to primary culture can be used. The medium may be any medium containing components necessary for cell proliferation or differentiation, specifically, Eagle's Minimum Essential Medium (EMEM), Dulbecco's Modified Eagle's Medium (DMEM), α-MEM, Glasgow MEM (GMEM), IMDM, RPMI1640, Ham's F-12, MCDB medium, Williams' Medium E, Hepatocyte thaw medium, MSC dedicated medium, KBM ADSC medium, or a mixture of these. The medium may contain additives such as various growth factors, differentiation inducers, antibiotics, hormones, amino acids, sugars, and salts, as necessary. From the viewpoint of safety in clinical application, the medium is preferably a serum-free medium that does not contain serum components of humans or animals other than humans.

[0039] 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.

[0040] The culture time is not particularly limited and can be appropriately determined depending on the cell proliferation rate and the desired size of the spheroids. The culture time is preferably 4 hours to 30 days (4 hours to 720 hours), more preferably 1 day to 14 days (24 hours to 336 hours), and even more preferably 1 day to 7 days (24 hours to 168 hours).

[0041] It is preferable to degas the cell culture substrate before culturing cells. The method of degassing is not particularly limited, and general methods such as spraying, pipetting, shaking, heating and cooling, centrifugation, vacuum degassing, and ultrasonic treatment can be used.

[0042] The ratio of stem cells and vascular endothelial cells during co-culture is not particularly limited, but from the viewpoint of forming a sufficient vascular structure within the spheroid, the ratio may be 20:1 to 2:1 or 10:1 to 2:1. When co-culture is performed on a cell culture substrate containing a fluorinated polyimide resin, from the viewpoint of providing sufficient oxygen supply from the fluorinated polyimide resin and reducing the hypoxic state inside the spheroid, the ratio of stem cells and vascular endothelial cells is preferably 100:7 to 5:1, and more preferably 10:1 to 5:1. EXAMPLES

[0043] [Preparing AdSC] Frozen human adipose-derived stem cells (PT-5006, purchased from Lonza) were thawed in a 37°C thermostatic water bath and added to 9 mL of KBM ADSC-2 medium (basal medium, manufactured by Kohjin Bio Co., Ltd.) containing 5% FBS and 1% antibiotics. The medium was then centrifuged at 210×g for 5 minutes to remove the supernatant, and the remaining cells were dispersed in 10 mL of basal medium to obtain a cell suspension. Culture flasks (culture area: 225 cm 2 ) and place 1.0 × 10 cells in each flask. 6 The cell suspension and basal medium were added so that the ratio of cells was 30 mL of medium per flask, and the cells were cultured (expanded) in a 5% (v / v) CO2 incubator at 37°C. After culturing, the medium was removed from the flask, 5 mL of Accutase (registered trademark) (manufactured by Promocell) was added, and the flask was left to stand at room temperature for about 5 minutes to detach the cells. The detachment solution containing the cells was then collected, washed with 10 mL of PBS, and transferred to a tube. The tube was centrifuged at 210 × g for 5 minutes to remove the supernatant, and the remaining cells were suspended in 4 mL of KBM ADSC-2 medium containing 1% antibiotics. The number of cells was counted, and the concentration of the cell suspension was adjusted to 2.0 × 10 6 Adjusted to cells / mL.

[0044] [Preparation of HUVECs] Frozen human umbilical vein endothelial cells (C2517A, purchased from Lonza) were thawed in a constant temperature water bath at 37°C and then added to 14 mL of the dedicated medium EGM (registered trademark) BulletKit (registered trademark) (manufactured by Lonza) to obtain a cell suspension. 2 ) with a cell volume of 5.0 × 10 5 The cell suspension and special medium were added so that the ratio of cells / 14 mL of medium / flask was 14, and the cells were cultured in a 5% (v / v) CO2 incubator at 37°C. The next day, the entire medium was replaced and expansion culture was performed. After culture, the medium was removed from the flask, 5 mL of Accutase (Promocell) was added, and the flask was left at room temperature for about 3 minutes to detach the cells. The detachment solution containing the cells was then collected, washed with EGM BulletKit, and transferred to a tube. The tube was centrifuged at 210 × g for 5 minutes to remove the supernatant, and the remaining cells were suspended in 4 mL of KBM ADSC-2 medium containing 1% antibiotics. The number of cells was counted, and the concentration of the cell suspension was adjusted to 4.0 × 10 5 cells / mL, 8.0×10 5 cells / mL, or 1.6 x 10 6 Adjusted to cells / mL.

[0045] [Defoaming treatment] The culture vessel containing the medium was degassed as follows. First, about 1 mL of phosphate buffered saline (PBS) was added to the culture vessel and pipetted, and the cell culture vessel was left to stand in a 5% (v / v) CO2 incubator at 37°C for 15 minutes. After pipetting again, the PBS was aspirated with an aspirator. Then, 0.2 mL of KBM ADSC-2 medium containing 1% antibiotics was added to the culture vessel, and the culture vessel was left to stand overnight in a 5% (v / v) CO2 incubator at 37°C.

[0046] <Example 1> The medium was removed from the culture vessel after the degassing treatment, and AdSCs and HUVECs were seeded so that the amount of AdSCs was 1000 cells / hole and the amount of HUVECs was 50, 100, or 200 cells / hole. The culture vessel was left to stand in a safety cabinet for 15 minutes, and then placed in a 5% (v / v) CO2 incubator at 37°C and left to stand for 4 hours. Next, KBM ADSC-2 medium containing 1% antibiotics was added to the culture vessel, and the culture vessel was again placed in a 5% (v / v) CO2 incubator at 37°C and cultured for 3 days to produce spheroids. As the culture vessel, a culture vessel with a diameter of about 300 μm, 400 holes, circular openings and bottom, fluorinated polyimide on the bottom, and MPC coating except for the openings was used.

[0047] The spheroids cultured for 3 days were photographed under a microscope, and the particle size was measured by image analysis (n=100, image analysis was performed using Nikon's NIS Elements). Figure 1 shows a micrograph of each spheroid, and Figure 2 shows a graph of the average particle size of each spheroid. The particle size of both the spheroids consisting of AdSC only and the spheroids consisting of AdSC and HUVEC was about 200 μm. In addition, when the particle size results of the spheroids were subjected to a multiple comparison test by the Tukey-Kramer method (statistical analysis software JMP (registered trademark) version 14.3.0, SAS), it was found that the particle size of the spheroids containing HUVEC was smaller than that of the spheroids consisting of AdSC only under the condition of 200 cells / hole. This is thought to be because the cell viability of the cells in the spheroids containing HUVEC was low under the condition of 200 cells / hole.

[0048] <Example 2> As in Example 1, the spheroids cultured for 3 days were fixed by treating with 4% paraformaldehyde phosphate buffer for 15 minutes, embedded in paraffin, and thin sections were prepared. HUVECs were stained using anti-CD31 antibody (Abcam) as the primary antibody and DAB (Nichirei) as the dye, and the HUVEC content in the spheroids was measured by image analysis (n=100, image analysis was performed using WinROOF from Mitani Shoji Co., Ltd.). The area ratio was calculated from the CD31 positivity rate per spheroid. Meanwhile, dead cells in the spheroids were stained with TUNEL (Millipore), and the cell viability was calculated by image analysis and the following formula (n=90). Cell viability (%) = total area of ​​one spheroid (100%) - TUNEL positive area (%)

[0049] Figure 3 shows micrographs of stained spheroids, and Figure 4 shows graphs of the percentage of HUVECs and cell viability in each spheroid. As the number of HUVECs seeded increased, the percentage of HUVECs in the spheroids increased. In addition, regardless of the number of HUVECs seeded, the cell viability in the spheroids was 95% or higher. However, when multiple comparison tests were performed using the Tukey-Kramer method, the cell viability decreased as the number of HUVECs seeded increased.

[0050] <Example 3> As in Example 1, the spheroids cultured for 3 days were fixed by treating with 4% paraformaldehyde phosphate buffer for 15 minutes. HUVECs were stained using anti-CD31 antibody as the primary antibody and an antibody labeled with Alexa Fluor (registered trademark) 594 as the secondary antibody, and the cell nuclei were stained with DAPI. SCALEVIEW-S4 was added to the stained spheroids and incubated overnight at 37°C. After transparency, the spheroids were observed with a confocal laser microscope from the bottom side of the culture vessel.

[0051] (A), (B), and (C) in Figure 5 (seeding number of HUVECs was 100 cells / well) are images of the z-stack of HUVEC staining images viewed from the side, side, and top, respectively, and (D) in Figure 5 is an image of the z-stack of stained images of HUVECs and cell nuclei viewed from the bottom (bottom side of the spheroid). In (E) in Figure 5, the upper left is a horizontal cross-sectional image of the spheroid, and the upper right and lower left are vertical cross-sectional images of the spheroid cut along lines I and II, respectively. As shown in these images, multiple luminal structures (blood vessels) were observed extending vertically from the flat surface of the spheroid (i.e., the part attached to the culture vessel), and these luminal structures formed a dome-shaped shape as a whole. In addition, AdSCs were present to cover the blood vessels forming this dome-shaped shape.

[0052] Figures 10 and 11 are z-stacks of stained images of spheroids produced by co-culture of AdSCs and HUVECs (50 cells / hole in Figure 10, 200 cells / hole in Figure 11). As can be seen from the results shown in Figures 10 and 11, even in the spheroids produced by co-culture of AdSCs and HUVECs at 50 cells / hole, vascular structures were formed, and blood vessels were confirmed to extend from the bottom of the film. However, the 50 cells / hole co-cultured spheroids formed fewer vascular structures and had less HUVEC attached to the bottom of the film compared to the 200 cells / hole co-cultured spheroids.

[0053] <Example 4> Furthermore, the spheroids were observed in the same manner as in Example 3, except that laminin or type IV collagen was stained using an anti-laminin antibody (Abcam) or an anti-type IV collagen antibody (Abcam) as the primary antibody and an antibody stained with Alexa Fluor (registered trademark) 488 as the secondary antibody.

[0054] Figure 6 (A) and (B) are horizontal cross-sectional images of the spheroid. Figure 6 (A) is a stained image of HUVEC, laminin, and cell nuclei, and Figure 6 (B) is a stained image of HUVEC, type IV collagen, and cell nuclei (HUVEC seeding number: 100 cells / well). As shown in Figure 6, laminin and type IV collagen were observed between the blood vessels forming a dome-shaped shape and AdSCs. This result indicates that a vascular structure with a basement membrane is formed in the spheroid. This structure is similar to the vascular structure in vivo, suggesting that the spheroid is functionally useful.

[0055] <Example 5> As in Example 1, the intracellular hypoxia sensing probe LOX-1 (MBL Life Sciences) was added to the spheroids cultured for 3 days at a final concentration of 2 μmol / L and cultured for 1 day. LOX-1 is a reagent whose fluorescence intensity increases with a decrease in oxygen concentration. The spheroids were observed with a confocal laser microscope, and the hypoxic state in the spheroids was evaluated from the fluorescence intensity (n=50). The microscope photographing conditions were standardized for each sample.

[0056] Figure 7 shows the fluorescence intensity of each spheroid. Figure 9 shows the fluorescence photograph of the spheroid. As can be seen from the results shown in Figure 7, the fluorescence intensity of the spheroids prepared by co-culture of AdSC and HUVEC at 100 or 200 cells / hole was significantly lower than that of the spheroids prepared by culturing AdSC alone. It was found that the presence of a vascular structure reduces the hypoxic state inside the spheroid. No significant difference was observed when the number of HUVECs seeded was 100 cells / hole and 200 cells / hole. This is because the dome-shaped luminal structure is thought to contribute to the supply of oxygen to the inside of the spheroid. It is known that the area near the center of the spheroid is particularly hypoxic, but spheroids with a dome-shaped luminal structure have a dense vascular structure near the center of the spheroid. On the other hand, past research has shown that polyimide film has higher oxygen permeability than polystyrene film. In the results of this experiment, a tubular structure was adhered to the bottom surface of the film to form a dome structure. It is believed that the formation of such a structure allows oxygen to be supplied from the bottom surface of the film, which has high oxygen permeability, to the inside of the spheroids via the network structure, thereby reducing the hypoxic state inside the spheroids. [Explanation of symbols]

[0057] 10...dome section, 20...flat section, 30...vascular structure, 100...spheroid.

Claims

1. A method for producing a spheroid in which stem cells and vascular endothelial cells are aggregated, comprising the steps of: The spheroid has a shape including a dome portion and a flat portion, A plurality of vascular structures are formed from the flat portion along a direction perpendicular to the flat portion, The flat portion is adhered to a cell-adhesive cell culture substrate, It is a spheroid, The method includes a step of co-culturing stem cells and vascular endothelial cells on a cell-adhesive cell culture substrate, The cell-adhesive cell culture substrate is a cell culture substrate containing a fluorinated polyimide resin, The stem cells are human adipose-derived stem cells, The method, wherein the vascular endothelial cells are human umbilical vein endothelial cells.

2. The method of claim 1 , wherein the plurality of vascular structures are generally dome-shaped.

3. The method according to claim 1 or 2, wherein a basement membrane is formed around the outer periphery of the vascular structure.

4. The method according to any one of claims 1 to 3, wherein the medium in which the co-culture is carried out is a serum-free medium.

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