Methods for preparing cell cultures

Abstract

The present invention addresses the problem of providing: a cell culture scaffold from which a cell culture can be recovered without being destroyed after culturing cells; and a method for producing a cell culture.　The cell culture scaffold according to the present invention is provided with a base having a water-repellent surface and a cell adhesion molecule formed on the water-repellent surface of the base. In addition, the method for producing a cell culture according to the present invention includes a step for using the cell culture scaffold to culture cells on the surface of the cell adhesion molecule. The cell culture scaffold according to the present invention can allow cells to be recovered in a spheroid shape without being damaged.

Description

[Technical Field]

[0001] The present invention relates to a cell culture scaffold for culturing cells and a method for producing a cell culture (particularly a three-dimensional cell aggregate) using the cell culture scaffold. [Background technology]

[0002] Enzymatic methods are known to detach cell cultures from the scaffold after cell culture, but this method makes it difficult to recover the cell cultures without destroying the bonds between the cells. Another known method involves using a temperature-responsive polymer in a cell culture scaffold and decomposing the temperature-responsive polymer through heat treatment, thereby detaching and recovering the cell culture from the scaffold (see, for example, Patent Document 1, etc.). Furthermore, in regenerative medicine, there is a strong demand for recovering cultured cells as spheroids. Culturing cells into spheroids has been proposed in, for example, Non-Patent Document 1. In this case, a dot pattern of hydroxyapatite is formed on a polytetrafluoroethylene (PTFE) surface, and cells are cultured on it to form spheroids. Also known as cell carriers are hydrophobic substrates coated with extracellular matrices such as proteins and peptides (Patent Document 2). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2013-099282 [Patent Document 2] Japanese Patent Application Laid-Open No. 2016-214210 [Non-patent literature]

[0004] [Non-Patent Document 1] Tamura et al., "Study on apatite dot fabrication method for cell / spheroid arrays," Biomedical Engineering Symposium 2013 (Proceedings p.141), presented on September 20, 2013 Summary of the Invention [Problem to be solved by the invention]

[0005] The method of Patent Document 1 requires that the culture-detachment process be carried out under temperature control with a narrow margin, making temperature control difficult and size control difficult. Non-Patent Document 1 describes culturing cells in a spheroid form, but does not disclose any method for detaching them from the substrate. Patent Document 2 describes coating an extracellular matrix on a hydrophobic substrate, but does not attempt to form spheroids in the first place.

[0006] Conventionally, when attempting to culture and recover cells into three-dimensionally assembled spheroids, the following problems arose. (1) Even if spheroids are formed, they cannot be peeled off from the substrate without deterioration. (2) Only floating spheroids of non-uniform size can be produced. (3) It is not possible to create spheroids in which all of the constituent cells are induced to differentiate. (4) It is not possible to create spheroids in which all of the cells that make up the spheroids have the appropriate cell polarity. The above-mentioned prior art documents were unable to solve these problems and were unable to recover cell spheroids.

[0007] Therefore, an object of the present invention is to provide a cell culture scaffold that allows the cell culture to be recovered without destroying it after cell culture, and a method for producing a cell culture using the same. [Means for solving the problem]

[0008] In order to solve the above problems, the present invention has the following configuration. That is, the cell culture kit for spheroid formation according to the present invention comprises: a substrate having a water-repellent surface made of a fluororesin; The sheet is attached to the substrate, has one or more holes, and when exposed to a culture medium for a certain period of time, its adhesion to the substrate weakens and it can be peeled off from the substrate by pipetting. After the holes are filled with cell adhesion molecules, the sheet is peeled off from the substrate. L Materials Has, The cell adhesion molecule is at least one of fibronectin, vitronectin, matrigel, collagen, laminin, and a fragment of the laminin. . Furthermore, the method for producing a cell culture according to the present invention comprises the steps of: a step of culturing cells on the surface of the cell adhesion molecules using a substrate having a water-repellent surface made of a fluororesin and a cell culture scaffold comprising cell adhesion molecules formed on the water-repellent surface of the substrate; The method includes a step of applying a stimulus to the interface by pipetting to detach and recover the cell adhesion molecules, utilizing the fact that the adhesive force at the interface between the water-repellent surface of the substrate and the cell adhesion molecules decreases after a certain period of time has elapsed since the start of cell culture. fruit, The cell adhesion molecule is at least one of fibronectin, vitronectin, matrigel, collagen, laminin, and a fragment of the laminin. . [Effects of the Invention]

[0009] When cells are cultured using the cell culture scaffold of the present invention, the interface between the water-repellent surface of the fluororesin and the cell adhesion molecules remains firmly attached for a certain period after the start of cell culture, but the adhesiveness of the interface decreases after a certain period of time. By utilizing this change in adhesiveness over time, cell cultures can be levitated from the cell culture scaffold without disruption after culturing the cells to a certain stage. Furthermore, by forming cell adhesion molecules in a pattern, cell cultures can be recovered as three-dimensional cell aggregates that are assembled in three dimensions. Furthermore, a large number of these three-dimensional cell aggregates of approximately the same size can be recovered. Furthermore, the present invention has the advantage of eliminating the difficulty of temperature control, which is required when using temperature-responsive polymers, and of easily controlling the size.

[0010] Therefore, it is believed that three-dimensional spheroids can be formed by adding an induction factor to stem cells cultured in two dimensions, forming cell polarity, and then detaching the cells from the substrate at the appropriate time while maintaining this polarity using an operation such as pipetting that causes minimal damage to the cells. This will solve the above-mentioned problems that have traditionally been encountered when obtaining spheroids. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram showing the procedure for producing a template in a stamping method. [Figure 2] 1A to 1C are schematic diagrams showing the procedure for producing a microstamp in a stamping method. [Figure 3] FIG. 1 is a schematic diagram showing the procedure for transferring cell adhesion molecules using a microstamp. [Figure 4] FIG. 1 is a schematic diagram showing a procedure for forming a pattern of cell adhesion molecules by the sealing method. [Figure 5] FIG. 10 is a diagram illustrating another embodiment of the cell culture kit for spheroid formation. [Figure 6] FIG. 1 is a schematic diagram showing cell culture using a cell culture scaffold according to the present invention. [Figure 7] FIG. 2 is a plan view of a photomask used in the examples. [Figure 8] 1 shows photographs of cell cultures before detachment in Examples 1 to 4. [Figure 9] 1 shows photographs of cell cultures after detachment in Examples 1 to 4. [Figure 10] 1 shows photographs of cell cultures after culturing and detachment in Examples 5 to 9. [Figure 11] 1 shows photographs of cell cultures after culturing and detachment in Examples 10 to 13. [Figure 12] 1 shows photographs of cell cultures after culturing and detachment in Examples 14 to 18. [Figure 13] 1 shows photographs of cell cultures after culturing and detachment in Examples 19 and 20. [Figure 14]This is an actual photograph of a cell adhesion molecule pattern formed using the sealing method. [Figure 15] Photographs of spheroids produced on cell culture scaffolds prepared by the sealing method. DETAILED DESCRIPTION OF THE INVENTION

[0012] The cell culture scaffold and the method for producing a cell culture according to the present invention will be described in detail below, but the scope of the present invention is not limited to these descriptions, and modifications other than those exemplified below may be made as appropriate within the scope that does not impair the spirit of the present invention.

[0013] [Base material] The substrate in the present invention has a water-repellent surface made of a fluororesin. Examples of substrates having such a water-repellent surface include petri dishes, wells, plates, multi-plates, flasks, and the like, which are made of a material other than fluororesin and have a fluororesin layer coated on the surface thereof. As the substrate made of a material other than fluororesin, for example, an existing substrate made of a plastic such as polystyrene or glass can be used. It is only necessary to appropriately select a substrate from which the fluororesin layer does not easily peel off.

[0014] The method for forming the fluororesin layer on the substrate is not particularly limited, and the layer can be formed by applying the fluororesin layer onto the substrate by a known application method such as spin coating, dip coating, or spray coating, and then curing the layer by heating or the like.

[0015] Furthermore, unlike the above, a substrate made of a fluororesin, such as a petri dish, may be used in which a fluororesin is used as the material of the substrate, and such a substrate is also included in the "substrate having a water-repellent surface made of a fluororesin" of the present invention.

[0016] As the fluororesin, known fluororesins can be used, and preferred examples thereof include polytetrafluoroethylene, polychlorotrifluoroethylene, polyvinylidene fluoride, polyvinyl fluoride, perfluoroalkoxy fluororesin, tetrafluoroethylene-hexafluoropropylene copolymer, ethylene-tetrafluoroethylene copolymer, and ethylene-chlorotrifluoroethylene copolymer.

[0017] [Cell adhesion molecule] Examples of cell adhesion molecules include extracellular matrices such as collagen, vitronectin, fibronectin, laminin, elastin, heparan sulfate, and proteoglycan, as well as fragmented molecules of their cell adhesion sites, and cationic polymers such as polylysine, polyethyleneimine, and polyallylamine hydrochloride. Such cell adhesion molecules themselves are conventionally known and can be appropriately selected depending on the type of cells to be cultured.

[0018] [Cell culture scaffold] The cell culture scaffold of the present invention comprises a substrate having the above-described water-repellent surface and cell adhesion molecules formed on the water-repellent surface of the substrate.

[0019] The cell adhesion molecules can be formed on the water-repellent surface of the substrate by known coating methods such as spin coating, dip coating, and spray coating. Alternatively, instead of simply coating, the cell adhesion molecules may be formed in a pattern on the water-repellent surface of the substrate.

[0020] Here, we will explain the term "patterned." A region where cell adhesion molecules are continuously formed on a substrate having a water-repellent surface is called a cell adhesion molecule region. The cell adhesion molecule region has an edge that forms a boundary between the cell adhesion molecule region and the substrate surface. In other words, the edge is the boundary between the cell adhesion molecule region and the substrate surface. The term "patterned" refers to a state in which at least one, preferably multiple, cell adhesion molecule regions are formed on a substrate. The cell adhesion molecule regions do not have to have the same shape and may differ in size. The cell adhesion molecule regions are also simply referred to as cell adhesion molecules.

[0021] When cell adhesion molecules are formed in a pattern, cultured cells can be collected as three-dimensional cell aggregates. Here, a three-dimensional cell aggregate is a three-dimensional collection of cells. A three-dimensional cell colony is called a spheroid. An organ-specific collection of cells consisting of multiple types of cells is called an organoid. These three-dimensional cell aggregates have been shown to exhibit tissue functions not seen in two-dimensional cell cultures, and are expected to be used in a variety of ways, such as to confirm the effects of drugs without using the human body. However, it has traditionally been difficult to recover three-dimensional cell aggregates without destroying them. Therefore, the present invention is extremely effective as a means of recovering three-dimensional cell aggregates such as those described above without destroying them, and also has the advantages of eliminating the difficulty of temperature control that occurs when using temperature-responsive polymers and of easy size control.

[0022] For example, by forming a pattern of cell adhesion molecules in the form of dots (approximately circular shapes), it is possible to form three-dimensional cell aggregates whose size depends on the size of each dot. In this case, the size of the dots (cell adhesion molecule regions) can be, for example, 100 to 1000 μm in diameter. If the cells to be cultured are about 5 to 25 μm in diameter, if the cell adhesion molecule regions are too small, the spheroids will be too small and insufficient for tissue cell formation. On the other hand, if the adhesion molecule regions are too large, the cells inside will die. It goes without saying that the present invention is not limited to the dot pattern, and various other pattern shapes can be adopted depending on the purpose.

[0023] The method for forming a pattern of cell adhesion molecules is not particularly limited, but examples include a method using an inkjet printer, a stamp method, a seal method, etc. The stamp method is preferable because it allows size control on the order of micrometers.

[0024] Furthermore, in the sealing method, by using a sealing material that has through holes in the shape of the cell adhesion molecule pattern (size, thickness) in advance on the substrate, it is possible to easily and accurately form cell adhesion molecule patterns without requiring proficiency in the technique to form the pattern of the cell adhesion molecules. A pattern of cell adhesion molecules can also be formed by attaching a sealant having through-holes in the pattern of cell adhesion molecules to a substrate, applying the cell adhesion molecules, and then peeling off the sealant.

[0025] The procedure for the stamping method will be described below with reference to FIGS. FIG. 1 shows the procedure for making a mold. Specifically, first, a resist 12 is applied onto a silicon substrate 11 (FIG. 1(a)). After the resist 12 is heated and cured with a heater 13 (FIG. 1(b)), it is exposed to light through a photomask 15 using a light source 14 to form a pattern (FIG. 1(c)). After that, it is developed and washed to obtain a mold 10 (FIG. 1(d)).

[0026] FIG. 2 shows the procedure for producing a microstamp using the above template. Specifically, first, a mold 10 (FIG. 2(a)) is placed in an outer container 9, and a rubber tube 21 is placed thereon (FIG. 2(b)). The mold 10 is then subjected to a release treatment using a release agent 22 (FIG. 2(c)). A stamp material 23 such as polydimethylsiloxane (PDMS) is then poured into the mold (FIG. 2(d)). This is then heated and cured in a constant-temperature dryer 24 (FIG. 2(e)), yielding a microstamp 20 (FIG. 2(f)). The microstamp 20 has protrusions 20a.

[0027] Next, the procedure for transferring cell adhesion molecules using the microstamp is shown in Figure 3. Figures 3(a), 3(c), and 3(e) show the flow of the procedure, while Figures 3(b), 3(d), and 3(f) show side views of Figures 3(a), 3(c), and 3(e), respectively.

[0028] Referring to Figure 3, first, a substrate 32 whose surface 32a has been treated to be water-repellent is prepared (Figures 3(a) and 3(b)). Cell adhesion molecules 20b are applied to the surface of the protrusions 20a of the microstamp 20. The protrusions 20a of the microstamp 20 are then brought into close contact with the water-repellent surface 32a of the substrate 32 (Figures 3(c) and 3(d)). This allows the pattern of the protrusions 20a of the microstamp 20 to be transferred to the water-repellent surface 32a of the substrate 32 as a pattern of cell adhesion molecules 20b (Figures 3(e) and 3(f)).

[0029] Next, we will explain the sealing method. Figure 4 shows the procedure for patterning cell adhesion molecules using the sealing method. Figures 4(a), 4(c), and 4(e) show the flow of the procedure, while Figures 4(b), 4(d), and 4(f) show side views of Figures 4(a), 4(c), and 4(e), respectively. The sealant 50 having holes 50a formed therein is placed in close contact with the substrate 32 (FIG. 4(a)). Here, "close contact" refers to a state in which no air is trapped between the sealant 50 and the substrate 32. Note that "no air trapped" refers to a state in which no air is trapped that would affect the pattern formation of cell adhesion molecules; for example, air bubbles that are sufficiently small relative to the film thickness of the sealant 50 may be present. The method of closely arranging the sealant 50 is not particularly limited. The sealant 50 may be applied to the entire surface of the base material 32 and then the holes 50a may be formed therein, or the sealant 50 with the holes 50a formed therein in advance may be attached to the base material 32.

[0030] Next, cell adhesion molecules 20b are placed on top of the sealant 50 (Figure 4(c)). The method for placing the cell adhesion molecules 20b is not particularly limited. Coating, dipping, spraying, etc. can be suitably used. Then, the cell adhesion molecules 20b on the sealant 50 are removed, and then the sealant 50 is removed, thereby obtaining a pattern of cell adhesion molecules 20b (Figure 4(e)).

[0031] In the sealing method, by controlling the thickness of the sealing material 50, the thickness 20bh of the resulting cell adhesion molecule 20b (cell adhesion molecule region) can be easily controlled. The sealant 50 must have the property of not leaving any components on the substrate 32 when it is peeled off from the substrate 32. If any components remain on the substrate 32, they may become contaminants for the cells being cultured.

[0032] Furthermore, the sealant 50 must have the property of not deteriorating the substrate 32 itself after being peeled off from the substrate 32. The sealant 50 must also have the property of being easily peelable from the substrate 32 and not peeling the fixed cell adhesion molecules 20b from the substrate 32 when being peeled off.

[0033] There are no particular limitations on the material of the sealing material 50 as long as it is a material that can achieve these properties, but suitable materials include resin materials such as thermosetting resins, thermoplastic resins, and photocurable resins, peptide materials such as gelatin, paper, clay (a mixture of inorganic materials and resin), etc.

[0034] Furthermore, adhering the sealant 50 to the substrate 32 requires a high level of skill. Therefore, a cell culture kit in which the sealant 50 with holes 50a formed on the substrate 32 is already formed is effective. The cell adhesion molecules 20b can be made from a material of the user's choice. Such a cell culture kit can be called a cell culture kit for spheroid formation in which the sealant 50 with holes 50a formed on the substrate 32 is closely attached to the substrate 32 having a water-repellent surface 32a. Of course, the cell culture kit may also include a kit in which cell adhesion molecules 20b are disposed in the holes 50a. Such a cell culture kit can be said to be a cell culture kit for spheroid formation in which a sealant 50 having holes 50a formed on a substrate 32 is formed and cell adhesion molecules 20b are disposed in the holes 50a.

[0035] Furthermore, in a cell culture kit for spheroid formation, it is preferable that the holes 50a formed in the sealing material 50 are arranged in an array of holes 50a of different sizes but similar shapes. In the cell culture scaffold according to the present invention, the quality of the spheroids formed by the cultured cells may vary depending on the size of the cell adhesion molecule region. Therefore, if a sealing material 50 having holes 50a of different sizes formed therein is placed on the substrate 32, it is easy to determine the size of the cell adhesion molecule region that should be formed when culturing cells for the first time.

[0036] 5(a) shows an example of a cell culture kit for spheroid formation in which holes 50a of different sizes are formed. The substrate 32 is located behind the sealing material 50. The holes 50aa, 50ab, 50ac, and 50ad are arranged in order of decreasing size. Of course, there are no limitations on the arrangement or size of the holes 50a.

[0037] Furthermore, the sealing material 50 may have a protrusion or tab at the edge for gripping during peeling to make it easier to peel off. Figure 5(b) shows a cell culture kit for spheroid formation with a protrusion 52 formed thereon. The substrate 32 is located behind the sealing material 50. The protrusion 52 is a portion that protrudes from the sealing material 50 in the same plane.

[0038] Moreover, Fig. 5(c) shows a cell culture kit for spheroid formation provided with tab 54. Fig. 5(d) is a side view of the cell culture kit for spheroid formation of Fig. 5(c). The tab 54 is a film sandwiched between the base material 32 and the sealing material 50. When peeling off the sealing material 50, the sealing material 50 can be peeled off from the base material 32 by pinching and pulling the tab with tweezers or the like.

[0039] [Cell culture] The cell culture will be described below with reference to FIG. When cells are cultured in a culture medium 41 using the cell culture scaffold of the present invention, the cells grow on the surface of the cell adhesion molecules 20b patterned on the water-repellent surface 32a of the substrate 32, as shown in Figure 6. Cell culture may be carried out by a conventional method, except that the cell culture scaffold according to the present invention is used.

[0040] In the early stages of cell culture, the interface between the water-repellent surface 32a and the cell adhesion molecules 20b has adhesive properties that prevent them from easily peeling off, but the adhesive properties of the interface between the water-repellent surface 32a and the cell adhesion molecules 20b decrease over time.

[0041] The cell adhesion molecules 20b naturally detach due to the decrease in adhesive strength. Alternatively, the cell adhesion molecules 20b can be detached by applying a stimulus to the interface where adhesive strength has decreased, such as by pipetting.

[0042] When the cell adhesion molecules 20b are detached, the cell culture 42 floats in the culture medium 41, and by collecting this, the cell culture 42 can be collected without being destroyed. In this case, the cell adhesion molecules 20b are surrounded and float integrally within the cell culture 42, as shown in FIG. By culturing stem cells on the cell adhesion molecules 20b and adding an inducer before the cell adhesion molecules 20b are detached from the substrate 32, it is possible to recover cells differentiated from the stem cells as spheroids.

[0043] The cells applicable to the present invention are basically assumed to be adhesive cells. Although not particularly limited, examples include epithelial-like cells, mesenchymal cells, fibrous cells, etc. Also included are pluripotent stem cells such as ES cells and iPS cells, tissue stem cells, progenitor cells, etc. [Example]

[0044] The present invention will be described in detail below using examples, but the present invention is not limited to these examples.

[0045] [Preparation of cell adhesion molecules and cultured cells] The cell adhesion molecules and cultured cells used in each example are described below. <Cell adhesion molecules> (1) Fibronectin (5 μg / cm 2 ): Fibronectin solution, derived from human plasma (063-05591 / Wako) (2) Vitronectin (1 μg / cm 2 ):rhVTN(220-02041 / Wako) (3) Matrigel (250-340 μg / ml): Corning Matrigel matrix, GFR (354230 / Corning) (4) Collagen (1.6 μg / ml): Collagen type IV from bovine lens (acid extracted) (0.5 mg / ml) (ASC-4-104-01 / Nippi)

[0046] <Cultured cells, source, and model number> (1) Hela: Tohoku University Institute of Development, Aging and Cancer Medical Cell Resource Center, TKG0331 (2) HepG2: Tohoku University Institute of Development, Aging and Cancer, Medical Cell Resource Center, TKG0205 (3) A549: Tohoku University Institute of Development, Aging and Cancer, Medical Cell Resource Center, TKG0184 (4) MCF7: Tohoku University Institute of Development, Aging and Cancer, Medical Cell Resource Center, TKG0479 (5)293::RIKEN BioResource Center, RCB1637 (6) iPS 409B2: RIKEN BioResource Center, HPS0076

[0047] <Culture conditions for each cell> (1)iPS cells (1-1) Seeding density 28.5 colonies / cm 2(Calculated at 500 cells / colony), or 1.5 x 10 for a single cell 5 cells / cm 2

[0048] (1-2)Culture solution A mixture of (a):(b):(c):(d):(e) = 100 mL:390 mL:5 mL:5 mL:3.5 μl was used. However, in the above, (a) to (e) are respectively: (a) SSR (191-18375 / Wako) (b)DMEM-F12(046-32275 / Wako) (c) 200 mM L-alanyl-L-glutamine (01102-82 / Nacalai) (d) MEM non-essential amino acid solution (16224004 / Nacalai) (e) 2-mercaptoethanol (135-07522 / Wako) is.

[0049] (1-3) Stripping solution A mixture of (a):(b) = 0.25 g:375 mL was used. However, in the above, (a) and (b) are respectively: (a) Dispase II (38302281 / Wako) (b)DMEM-F12(046-32275 / Wako) is.

[0050] (1-4) Cultivation procedure The detailed culture procedure is shown below.

[0051] (1-4-1) Reagents Dulbecco's modified Eagle's medium (DMEM; NISSUI 05919) D-MEM / Ham's F-12 (DMEM-F12; Wako 04230795) Stem Sure Replacement (SSR) (Wako 04632275) Fetal bovine serum (FBS; BioWest) MEM Non-Essential Amino Acid Solution (100x) (Nacalai 06344-56) 200mM L-alanyl-L-glutamine (powder; Nakarai 01102-82) 2-Mercaptoethanol (Wako 13706862) Mitomycin C (Wako 139-18711) Ethanol (Nacalai 14713-95) Ethylene glycol (Nacalai 058-00986) PBS (Ca, Mg free) (Nacalai 07269-84) Gelatin, Type B, Powder, Bioreagent for Cell Culture (Sigma G9391-100G) Trypsin powder (Nacalai 35547-64) EDTA (Nacalai 15105-35) Dispase II (Wako 38302281) Dimethyl sulfoxide (DMSO; Wako 043-07216) Acetamide (Nacalai 00117-32) Propylene glycol (Nacalai 29218-35) Bovine serum albumin (BSA; Nakarai 01281-84) CHAPS (Wako 34104721) Basic fibroblast growth factor (bFGF; Peprotech AF-100-18B) Y-27632 (Cayman 29218-35) Cell Reservoir One (Nacalai 07485-44)

[0052] (1-4-2) Preparation of reagents (1-4-2-1) L-glutamine solution Dissolve 3.212 g of L-glutamine in ultrapure water to obtain 110 ml of solution. After dissolving, sterilize by filtration using a 0.22 μm filter, dispense 10 ml into 15 ml centrifuge tubes, and store at -20°C.

[0053] (1-4-2-2)10%NaHCO3 solution Dissolve 10 g of NaHCO3 in ultrapure water to obtain 100 ml of a 10% NaHCO3 solution. After dissolving and sterilizing in an autoclave, store at room temperature.

[0054] (1-4-2-3) DMEM medium Dissolve 4.75 g of Dulbecco's Modified Eagle Medium (DMEM) in ultrapure water to obtain 500 ml of solution. Mix with a stirrer for 30 minutes, autoclave, and store.

[0055] (1-4-2-4) 0.1% gelatin solution Dissolve 0.5 g of gelatin in ultrapure water to obtain 500 ml of solution. After dissolving and sterilizing in an autoclave, store at room temperature.

[0056] (1-4-2-5)MMC solution Prepare a solution with the following composition: Mitomycin C (MMC) 10mg 1ml ethanol 9ml ethylene glycol After dissolution, the solution is sterilized by filtration using a 0.22 μm filter to obtain a solution.

[0057] (1-4-2-6) Dispase solution (200 PU / ml) Prepare a solution with the following composition: Dispase II 0.25g DMEM 150ml After dissolving and sterilizing by filtration using a 0.22 μm filter, dispense 1 ml into 15 ml centrifuge tubes and store at -20°C (200 PU / ml). When using, add 9 ml of DMEM to dilute 10 times.

[0058] (1-4-2-7) Culture medium for human iPS cells Prepare a medium with the following composition: DMEM-F12 390ml Non-essential amino acids 5ml 200mM L-glutamine 5ml SSR (20%) 100ml 2-mercaptoethanol (0.1 mM) 3.5 μl After preparation, store at 4°C and use within 2 weeks.

[0059] (1-4-2-8)basic FGF solution Dissolve the solvent in the following proportions, sterilize by filtration using a 0.22 μm filter, and prepare a solution. BSA 0.05g CHAPS 0.01g D-PBS(-) 10ml

[0060] (1-4-2-9) Cryopreservation solution DAP213 Prepare 2M DMSO, 1M acetamide, 3M propylene glycol / human iPS cell medium (DAP213) according to the following procedure. Dissolve 0.59g of acetamide in approximately 6ml of iPS cell culture medium. Sterilize by filtration using a 0.22µm filter and transfer to a 15ml centrifuge tube. Add 1.42 ml of DMSO and 2.2 ml of propylene glycol. Dilute to 10 ml with medium, mix well, dispense 0.5 ml into cryotubes, and store at -80°C. Thawing and freezing can be done several times.

[0061] (1-4-3) Preparation of feeder cells (1-4-3-1) Thawing of STO or mouse embryonic fibroblasts (MEF) 1) Bring the 10% FBS / DMEM medium to room temperature. 2) Transfer 8 ml of 10% FBS / DMEM medium to a 15 ml conical tube. 3) Thaw the frozen cells in a 37°C water bath until they are just about completely thawed. 4) Using a 1 ml disposable pipette, add the medium and transfer it to the 15 ml tube prepared in advance. Wash the inside of the tube with 1 ml of fresh 10% FBS / DMEM medium and collect any remaining cells. 5) Centrifuge at 1,500 rpm for 3 to 5 minutes. 6) Aspirate the supernatant and add 10 ml of 10% FBS / DMEM medium. Pipette thoroughly to suspend the cells so that no clumps remain. 7) Transfer the entire cell suspension to a φ100 dish. 8) Culture overnight in a CO2 incubator at 37°C to allow adhesion and proliferation. 9) The next day, observe the condition of the cells under a microscope. Bring the 10% FBS / DMEM medium to room temperature. 10) Remove the medium from the dish and add 10 ml of 10% FBS / DMEM medium to the dish. 11) Culture in a CO2 incubator at 37°C until confluent.

[0062] (1-4-3-2) Mitomycin C treatment 1) Ensure that the cells are confluent. 2) Add mitomycin C to a φ100 dish to a final concentration of 10 μg / ml (add 100 μl of 1 mg / ml mitomycin C stock solution to 10 ml of 10% FBS / DMEM medium). Shake the dish thoroughly to distribute the solution evenly over the cells. 3) Leave the plate in a CO2 incubator at 37°C for 2-3 hours. 4) After 2-3 hours, remove the medium from the dish and wash three times with 5 ml or more of PBS(-). 5) Add 10 ml of fresh 10% FBS / DMEM medium. 6) Incubate overnight at 37°C in a CO2 incubator. 7) In this state, most cells are non-dividing. 8) Wash with 5 ml of PBS(-) and discard by suction. Then add 1 ml of 0.25% trypsin-EDTA and let stand at 37°C for 2.5-3 minutes. 9) Add 3 ml of 10% FBS / DMEM medium, pipette, and collect the cells in a 15 ml centrifuge tube. 10) Centrifuge at 1,500 rpm for 3-5 minutes. 11) Aspirate the supernatant, add 900 μl of Cell Reservoir One, dispense 200 μl into 2 ml freezing tubes, and freeze. (1.95 × 10 6 (cells / cell, when thawed can be sown on φ60 x 2 plates)

[0063] (1-4-3-3) Preparation of feeder cells 1) The day before, add 0.1% gelatin solution to the dish (the amount of gelatin solution to add depending on the size of the dish is shown in Table 1 below). 2) Leave the plate in a CO2 incubator at 37°C for at least 30 minutes. 3) Bring the 10% FBS / DMEM medium to room temperature and transfer 5 ml of the 10% FBS / DMEM medium to a 15 ml conical tube. 4) Count some of the cells. 5) Thaw the frozen cells in a 37°C water bath until they are just about completely thawed. 6) Using a 1 ml disposable pipette, add the medium and transfer it to the 15 ml tube prepared in advance. Wash the inside of the tube with 1 ml of fresh 10% FBS / DMEM medium and collect any remaining cells. 7) Centrifuge at 1,500 rpm for 3-5 minutes. 8) Aspirate the supernatant and add 1 ml of 10% FBS / DMEM medium. Pipette thoroughly to suspend the cells so that no clumps remain. 9) Cell concentration is 0.8-1.0×10 6 Calculate the amount of medium to add to achieve cells / ml. 10) Remove the gelatin solution and inoculate a calculated amount of the fine suspension into a φ60 dish. 11) Incubate overnight at 37℃ in a CO2 incubator.

[0064] [Table 1]

[0065] (1-4-3-4) Subculture method for human iPS cells 1) Prepare the feeder cells the day before. (The following reagent volumes are for one 60mm cell culture dish) 2) Bring the human iPS cell medium and dispase solution to room temperature. 3) Add 0.5 ml of dispase solution to the dish, ensure that the solution is evenly distributed over the entire cell surface, and then incubate at 37°C in a CO2 incubator for 3 minutes. 4) Observe the state of the cells under a microscope and remove the dispase solution. 5) Incubate at 37°C for 10 minutes. It is desirable that more than half of the colonies have formed small clumps around them and are beginning to detach from the feeder cells. 6) Add 1 ml of human iPS cell medium and detach the cells from the dish. Pipette up and down several times (up to 10 times) to break up the colonies to an appropriate size. A Gilson P-1000 is recommended. (The number of cells should be approximately 100 per iPS cell colony.) If the colonies become too small, the efficiency of re-attachment and proliferation will be greatly reduced, so dissociate the colonies while checking under a microscope. 7) Centrifuge at 700 rpm for 2 minutes and remove as much of the supernatant as possible. 8) Remove the medium from the feeder cell dish and add 3 ml of human iPS cell medium to each dish. 9) Add 1.5-2 ml of medium to the conical tube and gently suspend the cells. Avoid vigorous pipetting, as the iPS cells will already have formed colonies of approximately 100 cells. If large colonies are visible, gently press the tip of the pipette used to draw up the cell suspension against the bottom of the conical tube and gently expel the cell suspension to loosen the cells. Add 1 ml of human iPS cell suspension per feeder cell dish. Add bFGF to a final concentration of 15 ng / ml. (If necessary, add Y-27532 solution to a final concentration of 10 μM / ml.) 10) Observe the cells under a microscope. Shake the dish thoroughly to ensure that the iPS cell colonies are distributed throughout the dish. 11) Incubate overnight at 37℃ in a CO2 incubator. 12) The next day, observe the condition of the cells under a microscope and change the medium. 13) After that, change the medium every day. The cells will become confluent in 3-4 days.

[0066] (1-4-3-5) Cryopreservation method for human iPS cells 1) Prepare one φ60 dish of confluent human iPS cells. 2) Bring the human iPS cell medium and dispase solution to room temperature. Prepare liquid nitrogen and ice. Write the cell name, date, passage number, etc. on a cryopreservation tube (Nulgene #5000-1012) and keep it chilled on ice. 3) Thaw the frozen storage solution DAP213 and keep it on ice. 4) Remove the medium from the human iPS cell dish, add an appropriate amount of PBS(-) to wash, and discard. 5) Add 500 μl of dispase solution to the dish, ensure that the solution is evenly distributed over the entire cell surface, and then incubate at 37°C in a CO2 incubator for 3 minutes. 6) Observe the state of the cells under a microscope and remove the dispase solution. 7) Incubate at 37°C for 10 minutes. 8) Add 3 ml of human iPS cell medium and detach the entire cell from the dish. 9) Collect the cells in a 15 ml centrifuge tube. 10) Centrifuge at 1500 rpm for 3-5 minutes and remove as much of the supernatant as possible. 11) Add 200 μl of DAP213 and gently suspend. Transfer to a previously prepared cryopreservation tube. Grasp the cryopreservation tube with tweezers and place in liquid nitrogen. DAP is highly cytotoxic, so work as quickly as possible. Ideally, within 15 seconds. 200 μl of DAP should be added regardless of the number of cells to be frozen. 12) Freeze in liquid nitrogen for 30 seconds to 1 minute until completely frozen. 13) Transfer to a liquid nitrogen storage container.

[0067] (1-4-3-6) Thawing human iPS cells 1) Prepare one φ60 dish of feeder cells per freezing tube of human iPS cells by the day before. 2) Transfer 10 ml of human iPS cell medium to a 15 ml conical tube and warm it in a 37°C water bath. 3) Add 1 ml of human iPS cell medium preheated to 37°C to the frozen human iPS cell freezing tube and pipette to rapidly thaw. Be sure to thaw and dilute quickly. Thawing in a water bath will significantly reduce cell viability after thawing. 4) After repeating the above procedure several times, collect the cell suspension in a 15 ml centrifuge tube and centrifuge at 1500 rpm for 3-5 minutes. 5) Aspirate and discard the supernatant, and add 1 ml of human iPS cell medium. Gently pipette the mixture up and down to suspend the iPS cell colonies so that they do not become too small. For large colonies, gently press the tip of the pipette that has sucked up the cell suspension against the bottom of the centrifuge tube to gently eject the cell suspension and loosen them. 6) Aspirate the feeder cell medium and add 3 ml of human iPS cell medium. 7) The entire volume is seeded and bFGF solution is added to a concentration of 15 ng / ml. (If necessary, add Y-27532 solution to a concentration of 10 μM / ml.) 8) Check the condition of the cells under a microscope. 9) Incubate overnight at 37℃ in a CO2 incubator. 10) The next day, check the condition of the cells under a microscope. Usually, many cells will be dead the day after thawing. Continue changing the medium once a day. Usually, the cells can be passaged after about three days.

[0068] (2) Cells other than iPS cells (2-1) Seeding density 7.8×10 4 cells / cm 2

[0069] (2-2)Culture solution A mixture of (a):(b) = 50:450 (mL) was used. However, in the above, (a) and (b) are respectively: (a) FBS (Biowest) (b) Dulbecco's modified Eagle's medium (DMEM; Nissui 05919) is.

[0070] (2-3) Stripping solution A mixture of (a):(b):(c) = 1.25 g:0.2 g:500 mL was used. However, in the above, (a) to (c) are respectively: (a) Trypsin (35547-64 / Nacalai) (b) EDTA (15105-35 / Nacalai) (c)PBS(-)(07269-84 / Nacalai) is.

[0071] (2-4) Cultivation procedure The detailed culture procedure is shown below.

[0072] (2-4-1) DMEM medium Dulbecco's Modified Eagle's Medium (DMEM; Nissui 05919): 433 ml (4.75 g dissolved in ultrapure water and autoclaved) 10% w / w NaHCO3 (Nacalai) solution: 7 ml (0.7 g dissolved in ultrapure water and autoclaved) 200mM L-glutamine (Nacalai) solution: 10ml (0.292g dissolved in ultrapure water and filtered) FBS (Biowest): 50ml (heat-inactivated)

[0073] (2-4-2) Trypsin / EDTA solution Trypsin (Nacalai): 1.25 g, EDTA (Nacalai): 0.2 g, PBS(-): 500 ml Dissolve, filter, dispense into 10 ml aliquots and store at -20°C

[0074] (2-4-3) Cultivation and subculture 1) Place the frozen cell line in a water bath at 37°C (500 μl of frozen solution). 2) Collect in 5 ml of 10 times the volume of DMEM medium and centrifuge at 1500 rpm for 3 minutes. 3) Discard the supernatant and seed the cells onto a φ60 or φ100 dish. 4) Culture in an incubator for several days. 5) Passage the cells as follows depending on the type of cultured cells (Hela, A549, HepG2, UV♀2, MCF7: 2-3 days, Hek293: 4-7 days).

[0075] Other than Hek293 1) Aspirate and discard the medium, add an appropriate amount of PBS(-), and aspirate and discard. 2) Add 500 μl of trypsin / EDTA solution to a φ60 tube or 1 ml to a φ100 tube and incubate at 37°C for 2.5-3 minutes. 3) Add DMEM medium in an amount at least twice the volume of the trypsin / EDTA solution, recover the cells, use several tens of μl to count the number of cells, and centrifuge the remainder at 1500 rpm for 3 minutes. 4) 3.0 x 10 in a 4-well dish 5 Seed cells in 1000 μL / well. 5) Culture in an incubator. Change the medium every two days.

[0076] Hek293 1) Aspirate and discard the medium, and add an appropriate amount of PBS(-). 2) Collect the cells by pipetting, use several tens of μl to count the number of cells, and centrifuge the remainder at 1500 rpm for 3 minutes. 3) 3.0 x 10 in a 4-well dish 5 Seed cells in 1000 μL / well. 4) Culture in an incubator. Change the medium every two days.

[0077] [Fabrication of Microstamps] The method for producing the microstamp used in each example will be described below. First, four types of templates were fabricated using the four types of photomasks shown in FIGS. 7(a) to (d). The dimensions of each photomask are summarized in the table below. The meanings of φ1 to φ3 and d are as shown in Figures 7(e) and (f), where φ1 is the diameter of the inner circle of the mask, φ2 is the diameter of the outer circle of the mask, φ3 is the diameter of each dot on the mask, and d is the distance between the centers of adjacent dots.

[0078] [Table 2]

[0079] The specific procedure for preparing the mold was as shown in Figure 1. Resist was applied to a silicon substrate, the resist was heated and solidified using a heater, and then the resist was exposed through each of the photomasks, developed, and washed to prepare the mold.

[0080] Next, four types of microstamps were fabricated using the four types of templates fabricated above. Specifically, the procedure was as shown in Figure 2 above, where the mold was treated with a release agent, polydimethylsiloxane (PDMS) was poured into it, and then dried using a constant temperature dryer to create a microstamp. Hereinafter, the microstamps will be referred to as microstamps 1 to 4, corresponding to photomasks 1 to 4 in Table 2 above.

[0081] [Experiment to verify the effects of the invention] Using the cell adhesion molecules and cells prepared as described above, various verification experiments were carried out as follows: In some examples, the microstamps prepared as described above were used.

[0082] Example 1 A fluororesin (Cytop CTX-809A, manufactured by AGC) was spin-coated onto a flat quartz substrate, which was then placed on a heater and baked at 200°C for 1 hour. Using microstamp 1 (φ3=250 μm), Matrigel was patterned as a cell adhesion molecule on the fluororesin layer according to the procedure shown in FIG. 3 described above. Next, cell culture and spheroid recovery were carried out according to the procedure shown in FIG. 6 described above. Specifically, the prepared cell culture scaffold was covered with culture medium and cells were seeded. The iPS cells prepared above were used and cultured in DMEM + 10% FBS medium at 37°C and 5% CO2. The number of cells seeded was 1.6 x 10 5 cells / cm 2 The cells were cultured for 10 days, and after 10 days, they were detached from the scaffold by applying a very weak stimulus by pipetting. The detached cells were observed under a phase-contrast microscope. Observation was performed every day or every other day, and the medium was changed every other day.

[0083] <Example 2> A cell culture scaffold was produced and cells were cultured in the same manner as in Example 1, except that Microstamp 2 (φ3=500 μm) was used instead of Microstamp 1.

[0084] Example 3 A cell culture scaffold was produced and cells were cultured in the same manner as in Example 1, except that Microstamp 3 (φ3=750 μm) was used instead of Microstamp 1.

[0085] Example 4 A cell culture scaffold was produced and cells were cultured in the same manner as in Example 1, except that Microstamp 4 (φ3=1000 μm) was used instead of Microstamp 1.

[0086] <Examples 5 to 20> Furthermore, experiments were carried out in accordance with the method of Example 1 using various combinations of cultured cells and cell adhesion molecules as shown in Table 3 below. However, in Examples 8 and 17 involving iPS cells, cell culture scaffolds were used in which various cell adhesion molecules were patterned on a fluororesin layer using Microstamp 2 (φ3 = 500 μm) or Microstamp 3 (φ3 = 750 μm).On the other hand, for other cells, cell culture scaffolds were used in which various cell adhesion molecules were patterned on a fluororesin layer using a pipette in the form of dots (diameters of approximately 1000 μm for fibronectin and vitronectin, and approximately 1500 μm for fibronectin and vitronectin).

[0087] [Table 3]

[0088] [Verification experiment results] Photographs of the cell cultures before detachment for each of Examples 1 to 4 are shown in Figure 8. Figure 8(a) is a photograph of the cell cultures before detachment for Example 1, Figure 8(b) is a photograph of the cell cultures before detachment for Example 2, Figure 8(c) is a photograph of the cell cultures before detachment for Example 3, and Figure 8(d) is a photograph of the cell cultures after detachment for Example 4. Figure 9(a) is a photograph of the cell cultures after detachment for Example 1, Figure 9(b) is a photograph of the cell cultures after detachment for Example 2, Figure 9(c) is a photograph of the cell cultures after detachment for Example 3, and Figure 9(d) is a photograph of the cell cultures after detachment for Example 4. As can be seen from Figures 8 and 9, in both Examples, cell cultures (spheroids) were formed, and it was found that these could be suspended in the culture medium and collected without being destroyed. It was also found that the size of the spheroids could be controlled depending on the dimensions of the dots.

[0089] Photographs of Examples 5 to 20 are shown in Figures 10 to 13. Initial adhesiveness, pattern formability, and spheroid formation were evaluated, and the results are shown in Table 3 above and Figures 10 to 13.

[0090] The spheroid formation was evaluated according to the following criteria. 〇: When the cell aggregate is large and thick enough to be considered spherical in three dimensions. △: When the cell aggregate has a certain thickness and size in three dimensions ×: Cells are barely aggregated and are dispersed two-dimensionally (If it is thick, the transmitted light will be reduced when observed under a microscope, making it appear darker in color.)

[0091] As shown in Table 3 and Figures 10 to 13, it was found that cell cultures (spheroids) were formed using various combinations of cultured cells and cell adhesion molecules, and that these could be suspended in culture medium and recovered without being destroyed.

[0092] [Sealing method] Figure 14 shows an example of a pattern of cell adhesion molecules 20b produced by the sealing method. The substrate 32 was a flat quartz substrate spin-coated with a fluororesin (CYTOP CTX-809A, manufactured by AGC Corporation). The sealing material 50 was formed by spin-coating an ultraviolet-curable resin (manufactured by DYMAX Corporation) onto the substrate 32, curing it with ultraviolet light using a negative dot pattern as a mask, and then removing the uncured portions. Figure 14(a) shows a sealing material 50 with four holes 50a formed on the substrate 32. The scale bar is 10 mm.

[0093] 14(b) shows the state in which the sealing material 50 has been peeled off from the base material 32. Even when the sealing material 50 was peeled off from the base material 32, no components of the sealing material 50 remained on the base material 32, and the sealing material 50 could be peeled off cleanly.

[0094] Figure 14(c) is a photograph of the patterned cell adhesion molecules 20b (cell adhesion molecule region) obtained by applying cell adhesion molecules 20b to a sealant 50 formed on a substrate 32, leaving the sealant 50 to set for one hour, and then peeling off the sealant 50. Cylindrical cell adhesion molecules 20b with a diameter of 1 mm and a thickness of 0.1 mm were obtained. The cell adhesion molecules 20b on the substrate 32 are the cell culture scaffold of the present invention. Furthermore, the substrate 32 in Figure 14(a) with the sealant 50 with holes 50a formed thereon and tightly attached thereto constitutes a cell culture kit for spheroid formation of the present invention.

[0095] Next, spheroids were generated using the cell culture scaffold prepared by the sealing method. First, the base material 32 with the sealing material 50 having holes 50a with a diameter of 1 mm (=1000 μm) formed therein shown in FIG. 14 was gas sterilized. Next, Matrigel was added onto the sealing material 50 (to arrange the cell adhesion molecules), and the mixture was allowed to settle in an incubator at 37°C for 1 hour.

[0096] The sealing material 50 was peeled off with flame-sterilized tweezers to obtain a cell culture scaffold patterned with cell adhesion molecule (Matrigel) dots having a diameter of 1000 μm. Next, the cells (Hek293) used in Examples 5, 10, and 14 were seeded on the substrate 32. Then, they were cultured using the same culture method as in the above Examples (the method described in "(2-4) Culture Procedure"). After seeding the cells, they were detached from the substrate 32 by pipetting on the fifth day, and the cells were observed on the sixth day.

[0097] The observation results are shown in Figure 15. Hek293 cell spheroids were obtained. [Industrial Applicability]

[0098] The cell culture scaffold according to the present invention can be suitably used to obtain spheroid cell cultures. [Explanation of symbols]

[0099] 9 Outer container 10 Mold 11 Circuit Board 12 Resist 13 Heater 14 Light source 15 Photomask 20 Microstamps 20b Cell adhesion molecules 20bh thickness 21 Rubber tube 22 Release agent 23 Stamp materials 24 Constant temperature dryer 20a Convex part 32 Base material 32a (water-repellent) surface 41 Culture solution 42 Cell culture 50 Sealing material 50a hole

Claims

1. a step of culturing cells on the surface of the cell adhesion molecules using a substrate having a water-repellent surface made of a fluororesin and a cell culture scaffold comprising cell adhesion molecules formed on the water-repellent surface of the substrate; the adhesive force at the interface between the water-repellent surface of the substrate and the cell adhesion molecules decreases after a certain period of time has elapsed since the start of cell culture, and the interface is stimulated by pipetting to detach and recover the cell adhesion molecules; A method for producing a cell culture, wherein the cell adhesion molecule is at least one of fibronectin, vitronectin, matrigel, collagen, laminin, and a fragment of the laminin.

2. 2. The method for producing a cell culture according to claim 1, wherein the cell adhesion molecule is an extracellular matrix molecule.

3. 3. The method for producing a cell culture according to claim 1, wherein the cell adhesion molecules are formed in a pattern on the water-repellent surface of the substrate.

4. 4. The method for producing a cell culture according to claim 1, wherein the cell adhesion molecules are formed in the shape of dots having a diameter of 100 to 1000 μm.

5. 5. The method for producing a cell culture according to claim 1, wherein the cells are adherent cells.

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