Microwell sheet

Abstract

This microwell sheet includes recessed microwells each formed to have an opening and a planar bottom. The depth from the opening to the bottom is at least 1.4 times the diameter of the opening.

Description

Microwell Sheet

[0001] The present invention relates to a microwell sheet for producing or placing cell aggregates, a gas-permeable microwell sheet, a cell culture method, and a method for evaluating drugs using the same. More particularly, the present invention relates to a microwell sheet in which the depth from the opening to the bottom of the microwell is 1.4 times or more the diameter of the opening, and the bottom is flat, as well as a cell culture method and a method for evaluating drugs using the same.

[0002] Spherical three-dimensional (3D) cell aggregates have various applications in regenerative medicine and cell-based assays due to their stability and high functionality. Conventional methods for forming aggregates include, for example, silicone resin culture vessels with circular openings and U-shaped bottoms (see Patent Document 1) and microwell-based culture systems with honeycomb-shaped openings and flat bottoms (see Non-Patent Document 1).

[0003] However, the well structure described in Patent Document 1 has a U-shaped bottom, which causes a liquid flow that pushes aggregates outward when culture medium enters the well. This results in aggregates easily coming out of the well or entering other wells. Furthermore, the honeycomb microwell sheet described in Non-Patent Document 1 is designed with shallow wells to facilitate cell handling, and therefore, aggregates can easily escape from the wells during culture medium exchange or perfusion, resulting in the same problems as in Patent Document 1.

[0004] Producing a large number of aggregates requires human or mechanical operation, which is time-consuming and labor-intensive. Moreover, the aggregates obtained in this way are often not of uniform quality, and the amount of aggregates obtained is low in terms of productivity per unit area or volume, making it difficult to form homogeneous aggregates.

[0005] Furthermore, in drug efficacy tests using biomimetic systems such as microphysiological systems (MPS), different types of cells, such as hepatocytes and cardiomyocytes, are sometimes co-cultured. However, during so-called perfusion culture, where the culture medium in a hepatocyte well is transferred to a cardiomyocyte well, cell aggregates in one well can be dislodged from the other well due to the flow of the culture medium, forming large aggregates that lead to cell death. In addition, the flow of these aggregates reduces the number of cells required for drug testing. This has resulted in problems in accurately evaluating drug efficacy.

[0006] Furthermore, even when used for cell proliferation, there was a problem where cell aggregates would jump out of the wells during culture medium changes, making it difficult to increase the number of cells as desired.

[0007] International public access number WO2024 / 190691A1

[0008] 2013 Combination of microwell structures and direct oxygenation enables efficient and size-regulated aggregate formation of an insulin-secreting pancreatic β-cell line(2014 Jan-Feb;30(1):178-87. doi: 10.1002 / btpr.1837.)

[0009] Given the above background, there was a need for a microwell sheet that could be used to manufacture or place cell aggregates without them spilling out of the container.

[0010] The inventors of the present invention have diligently studied to solve the above problems and have succeeded in manufacturing a microwell sheet, particularly a gas-permeable microwell sheet, thereby completing the present invention. That is, the present invention is as follows: [1] A microwell sheet having concave microwells having an opening and a planar bottom, wherein the depth from the opening to the bottom is at least 1.4 times the diameter of the opening. [2] The microwell sheet according to [1], wherein the shape of the opening surface and bottom surface of the opening and bottom are honeycomb-shaped. [3] The microwell sheet according to [1] or [2], wherein there is a wall of equal thickness between one adjacent microwell and another microwell. [4] The microwell sheet according to any one of [1] to [3], which is gas-permeable. [5] The microwell sheet according to any one of [1] to [4], wherein the thickness of the bottom is 0.8 μm or less. [6] The microwell sheet according to any one of [1] to [5], which is made of silicone resin. [7] A cell culture method comprising the step of forming spheroids or organoids by culturing cells in the microwells of a microwell sheet described in any of [1] to [6]. [8] A method for producing spheroids or organoids comprising the step of forming spheroids or organoids by culturing cells in the microwells of a microwell sheet described in any of [1] to [6]. [9] The method according to [7], wherein the culture is perfusion culture.

[10] The method according to [8], wherein the culture is perfusion culture.

[11] A microphysiological system comprising a microwell sheet described in any of [1] to [6] and a container in which the microwell sheet is placed.

[12] A method for evaluating a drug comprising the step of culturing spheroids or organoids placed in the microwells of the microphysiological system described in

[11] while supplying gas, and introducing a drug to be evaluated into the container of the microphysiological system.

[0011] According to the present invention, as described above, it is possible to suppress the decrease in the number of aggregates (cells) that occurs when cells in a well leave the well and enter other wells, or when aggregates are washed away, due to culture medium exchange or perfusion culture. As a result, aggregates with a controlled number of cells can be obtained in each well, and can be used as a cell source during tissue formation. Furthermore, by making the material constituting the microwells gas permeable, such as oxygen permeability, it is possible to obtain aggregates that do not result in an increase in dead cells due to oxygen deficiency, a decrease in hepatocyte function, or a state where hepatocytes no longer function as hepatocytes, which can occur when creating aggregates of hepatocytes, for example.

[0012] Furthermore, in fields such as toxicity testing, for example, toxicity testing using hepatocyte aggregates in microwells, or experimental safety evaluation using animal experiments or primary human stem cells (PHH) as non-clinical drug tests, it can solve problems arising from differences in drug metabolism between experimental animals and humans, as well as various problems associated with using hepatocyte aggregates, such as lot-to-lot differences in PHH and difficulties in long-term culture.

[0013] This figure shows the microwell sheet of the present invention. This figure shows the microphysiological system of the present invention. This figure shows the microwell sheet according to an embodiment of the present invention. This is a cross-sectional view showing the depth from the opening to the bottom of the microwell according to an embodiment of the present invention. This is a micrograph showing the morphology of cells when iPS cells are cultured using microwell sheets with aspect ratios of 1:1.5 and 1:1. This is a micrograph showing the morphology of cells when HepG2 cells are cultured using microwell sheets with aspect ratios of 1:1.5 and 1:1. This is a micrograph showing the morphology of cells when HepG2 cells are cultured using the microwell sheet of the present invention and spheroids are collected from the microwells on the 5th day of culture. This is a micrograph showing the morphology of cells when HepG2 cells are perfused using microwell sheets with aspect ratios of 1:1 and 1:1.5 (at the start of perfusion culture). This is a micrograph showing the morphology of cells when HepG2 cells are perfused using microwell sheets with aspect ratios of 1:1 and 1:1.5 (24 hours after the start of perfusion). These are micrographs showing the cell morphology of HepG2 cells perfused using microwell sheets with aspect ratios of 1:1 and 1:1.5 (48 hours after the start of perfusion). These are micrographs showing the cell morphology of HepG2 cells perfused using microwell sheets with aspect ratios of 1:1 and 1:1.5 (72 hours after the start of perfusion). These are micrographs showing the cell morphology of HepG2 cells perfused using microwell sheets with aspect ratios of 1:1 and 1:1.5 (144 hours after the start of perfusion). This is a schematic diagram showing one of the component units of an on-chip pump type multi-organ MPS device used in perfusion culture.

[0014] The present invention relates to a microwell sheet in which a concave microwell having an opening and a flat bottom is formed. In the microwell sheet of the present invention, the distance (depth) from the opening to the bottom is at least 1.4 times the diameter of the opening, and the bottom is flat. Furthermore, the microwell sheet is gas permeable. The present invention also relates to a method for forming and culturing cell aggregates, such as spheroids and organoids, using these microwell sheets, and a method for evaluating drugs using these. In the present invention, the cell aggregates do not come out of the wells even when a flow rate is applied to the liquid culture medium. Embodiments of the present invention will be described below.

[0015] [Definitions] In this invention, the diameter of the opening refers to the diameter of the inscribed circle if the opening is polygonal. The aspect ratio refers to the ratio (b / a) of the length of the opening diameter (a) to the distance (depth) from the opening to the bottom (b). The wall thickness refers to the thickness (w) between one adjacent microwell (hereinafter also simply referred to as a well) and another well, and the bottom thickness refers to the distance (d) from the bottom surface of the well to the surface of the sheet opposite to the well-forming surface. Embodiments of this invention will be described below with reference to the drawings.

[0016] (Microwell Sheet) Figure 1 is a schematic diagram of the microwell sheet 1 of the present invention, where panel A is a plan view showing a part of the microwell sheet, and panel B is a cross-sectional side view of panel A along the line p1-p2. In Figure 1, the microwell sheet 1 has an opening D, and a plurality of microwells 10 (hereinafter also simply referred to as "wells") are arranged therein, with a depth of b from the opening to the bottom. The shape of the opening surface and the bottom surface of the wells 10 is circular or polygonal, and is not particularly limited. However, it is preferable that they be polygonal in order to form uniform wells on the sheet surface and to ensure that the wall thickness is also uniform. In Figure 1, a hexagon (honeycomb shape) is shown as a preferred embodiment, and the wall portions 11 separating adjacent wells 10 also have a uniform thickness (w). Furthermore, the bottom portion 12 that forms the bottom of the well has a thickness d, and it is preferable that its material is gas permeable. Furthermore, it is preferable that the diameter a1 of the opening D and the diameter a2 of the bottom portion have the same shape and the same length.

[0017] The material used to form the microwell sheet 1 is not particularly limited as long as the bottom portion 12 is gas permeable. Examples include silicone resins such as polydimethylsiloxane, crystalline polyolefin resins such as polymethylpentene, polytrimethylsilylpropyne, and natural rubber. When forming the microwell sheet 1 of the present invention, polydimethylsiloxane is preferably used in terms of its microprocessability and oxygen permeability.

[0018] In the present invention, the well 10 has an aspect ratio (b / a1) of 1.4 or more, which is the ratio of the diameter a1 of the opening D to the depth b of the well 10. The diameter of the opening D is appropriately selected according to the intended use of the well, but is, for example, in the range of 32 μm to 500 μm. For example, when the purpose is to produce cell aggregates, depending on the cells used, the desired cell aggregates can be formed by forming wells with a diameter in the range of 70 μm to 300 μm, preferably 100 μm to 200 μm, and particularly preferably around 126 μm. When the purpose is organoids, uniform organoids can be formed by forming wells with a larger size than the cell aggregates, for example, a diameter of 200 μm to 600 μm, preferably about 250 μm to 500 μm. In the case of gene systems such as gene aggregates, wells with a smaller diameter than the cell aggregates, for example, a diameter of 150 μm or less, preferably 32 μm to 126 μm, can be used.

[0019] If the aspect ratio is smaller than this range, the various aggregates formed in the wells may come out of the wells when the culture medium is changed or perfused, resulting in a loss of the cell source, or they may enter other wells, causing uneven aggregate size, which is undesirable. Also, if the aspect ratio is too large, the well formation rate decreases, it becomes difficult to remove cell aggregates and organoids, and when used in systems such as MPS for perfusing the culture medium, it may block the channel through which the culture medium passes, preventing perfusion, which is also undesirable. Therefore, the aspect ratio is preferably between 1.4 and 2, and particularly between 1.5 and 1.8.

[0020] Furthermore, in the present invention, it is preferable that the shape of the opening surface of the opening D and the shape of the bottom surface of the bottom 12 (the bottom 12 inside the well) are the same. This makes it easier to form wells with the same diameter from the opening D to the bottom 12. If the shape of the opening surface of the opening D and the shape of the bottom surface of the bottom 12 are different, the internal shape will either widen in a tapered manner towards the opening or narrow. When cells are placed in such a well, if it widens towards the opening, the cells are more likely to jump out, and if the opening narrows, the cell aggregates will become larger than the opening inside the well, making it difficult to remove the formed cell aggregates. In the present invention, it is preferable to make the shape of the opening surface of the opening D and the shape of the bottom surface of the bottom 12 the same, and to form wells so that each surface is parallel, as this makes it easier to make the cell morphology uniform and makes it easier to remove the cells from the well.

[0021] Furthermore, in this invention, the shape of the bottom 12 (the shape within the well 10) must be flat. If the shape of the bottom 12 is U-shaped or spherical, cell aggregates are more likely to form, but cell sources are more likely to escape from the well due to convection of the culture medium during culture medium exchange. Also, depending on the shape, oxygen-containing gases may not enter the well uniformly, making it difficult to adjust the gas concentration to the level required for various cells, which is undesirable.

[0022] In Figure 1, the thickness d of the bottom 12 is 0.8 μm or less, preferably in the range of 0.6 to 0.2 μm. If it is thinner than this range, the strength of the bottom becomes too low, causing it to break during various operations and the culture medium and cells to escape from the bottom. If it exceeds this range, the permeability of various gases, including oxygen, decreases, making it difficult to supply the gases necessary for the cells. From the viewpoint of bottom strength and gas permeability, the particularly preferred thickness d of the bottom 12 is in the range of 0.5 to 0.2 μm. Furthermore, when using a microwell sheet placed on the surface of other oxygen-permeable materials such as polymethylpentene, it is important to consider the oxygen permeability of the oxygen-permeable material used and set the thickness in the range of 0.1 to 0.5 μm.

[0023] Furthermore, the thickness w of the wall portion 11 is preferably in the range of 30 to 130 μm when the well diameter is 200 μm or less, and preferably in the range of 80 to 150 μm when the well diameter exceeds this, from the viewpoint of maintaining the shape of the microwell sheet, ease of handling, and preventing warping. If it is thinner than this range, the number of wells can be increased, but the wells will warp, causing the well shape to deform during use. As a result, it becomes impossible to accurately place the number of cells and the amount of culture medium into the wells, making it difficult to obtain uniform cell aggregates. Also, when using the microwell sheet 1 of the present invention as a cell culture plate, if it does not maintain its shape or warps when attached to the plate frame, it cannot be attached accurately, which is undesirable because it can cause leakage of culture medium or changes in gas concentration, making uniform cell culture impossible. From the viewpoint of the number of wells and ease of handling, the thickness of the wall portion 11 is particularly preferably in the range of 30 to 120 μm.

[0024] The microwell sheet 1 of the present invention can also be surface-treated to prevent cells from adhering to the wells when forming cell aggregates or organoids. Surface treatments include coating with polymers or Pluronic surfactants that have characteristics not recognizable by cells, such as PEG (polyethylene glycol), MPC (2-methacryloyloxyethyl phosphorylcholine), and PMEA (poly(2-methoxyethyl acrylate)). Alternatively, cell-specific recognition materials that are recognized only by certain cells, such as fusion proteins formed by conjugating antibody Fc to various cadherins like E-Cadhein-Fc, N-Cadherin-Fc, and VE-Cadherin-Fc, or poly-N-vinylbenzyl-D-lactoneamide (PVLA), can be used. The treatment method involves dissolving these materials in a solvent to a specific concentration and immersing the microwell sheet in the solution, or placing the solution in the wells to be treated and removing them from the solution or from the wells after a specific time has elapsed, thereby performing cell non-adhesion treatment.

[0025] (Formation of Spheroids and Organoids) Cell aggregates can be formed by introducing specific cells or stem cells into the wells of the microwell sheet and performing cell culture. Alternatively, pluripotent stem cells such as iPS cells or ES cells can be cultured in the wells, and then differentiation-inducing agents such as growth factors can be added to form desired aggregates such as endoderm, ectoderm, or mesoderm. Furthermore, to form desired tissues (organs) within the wells, the culture can be continued under culture conditions such as culture medium and various additives to form organoids. The culture medium can be, but is not limited to, media commonly used for culturing animal cells, such as MEM, DMEM, RPMI-1640, Ham's F-12, DMEM / F-12, and Essential 8 (registered trademark). Culture can be performed in an incubator, for example, at 37°C and under a 5% CO2 atmosphere.

[0026] By using the microwell sheet of the present invention, spheroids and organoids can be cultured by perfusion instead of changing the culture medium each time. Normally, when culture medium is perfused, cells in the wells may escape from the wells and disappear, or cells may enter other wells, making it difficult to control cell morphology and cell number. However, by using the microwell sheet of the present invention, cells do not escape from the wells even in perfusion culture, enabling stable culture.

[0027] The present invention provides a microphysiological system (MPS) 2 comprising a microwell sheet and a container for arranging the microwell sheet. The microwell sheet of the present invention can be used as a biomimetic system by means of the MPS.

[0028] Figure 2 is a schematic diagram showing MPS2. In Figure 2, MPS2 comprises a container 21a equipped with the microwell sheet 1 of the present invention, and a container 21b connected to the container 21a by a connecting part (connecting tube) 25. In the container 21a in Figure 2, the microwell sheet 1 of the present invention is placed on the bottom surface of the container 21a, and cell aggregates 23 are formed in the wells of the microwell sheet 1. The MPS2 is filled with culture medium 24, which flows in from the inlet 22a of the container 21a, perfuses the container 21a, and flows into the container 21b through the connecting part 25 that connects the container 21b. The culture medium 24 that has flowed into the container 21b is perfusated and discharged from the outlet 22b.

[0029] In the present invention, the above-mentioned MPS2 can be used to perform drug efficacy tests and the like. For example, consider an evaluation system for the degradation of drugs by liver cell aggregates. In Figure 2, the necessary cell aggregates are formed in the microwells of the microwell sheet 1. For example, in a drug efficacy test, liver cells are seeded in the wells and cultured to form liver cell aggregates (cell aggregates 23), and then perfused while supplying oxygen. Subsequently, the drug is added to the culture medium 24, the drug is degraded by the liver cell aggregates in the wells, and metabolites 27 from the degraded liver cell aggregates are moved to the container 2 along with the culture medium and transported to the next cell 26, making it possible to evaluate how the metabolites 27 affect that cell 26.

[0030] By using the microwell sheet 1 of the present invention in MPS2, the function of hepatocytes can be maintained and improved, and the number of cells can also be increased, making it possible to evaluate drugs in a manner closer to that of a living organism. The MPS of the present invention is not limited to two containers; it is also possible to connect the next container (not shown) to the outlet 22b of container 21b and continuously evaluate drugs, etc.

[0031] The invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0032] [Preparation of microwell sheet] Mold production: A SU-8 negative photoresist (manufactured by MicroChem) was spin-coated on a clean silicon wafer to a thickness of (i) 190 µm or (ii) 130 µm, and soft-baked. Subsequently, each SU-8 layer on the silicon wafer was irradiated with ultraviolet (UV) light from the upper surface of a glass photomask, and developed using an SU-8 developer (manufactured by MicroChem). The photomask was produced by drawing the honeycomb pattern (inscribed circle diameter: 126 µm) shown in Figure 3 on a mask blank (CBK400Du-AZP, manufactured by Clean Surface technology) using a laser pattern generator (DWL200, manufactured by HEIDELBERG INSTRUMENTS). To facilitate peeling of the microwell sheet from the surface of the patterned SU-8, CHF₃ coating was performed using a reactive ion etching system (model RIE-10NR, manufactured by SAMCO), and molds for microwell sheets were produced, wherein the ratio of depth to the diameter of the opening or bottom (aspect ratio, i.e., "depth" / "diameter of the opening or bottom") is 1 (bottom length (diameter) : wall height = 1:1) and 1.5 (bottom length : wall height = 1:1.5). Hereinafter, when the aspect ratio is expressed as X:Y, it represents "bottom length : wall height".

[0033] Production of microwell sheet: A polydimethylsiloxane (PDMS) solution containing a prepolymer and a catalyst (Silpot184, manufactured by Dow Corning) at a ratio of 9:1 (w / w) was poured into the mold, heated at 75°C for 2 hours, and then peeled from the mold to produce a honeycomb-shaped microwell sheet with an aspect ratio of 1:1 and a honeycomb-shaped microwell sheet with an aspect ratio of 1:1.5 shown in Figure 3 and Figure 4.

[0034] [Preparation of Microwell Plates] PDMS microwell sheets with honeycomb-shaped wells having an aspect ratio of 1:1 and 1:1.5 for the inscribed circle diameter and depth, respectively, were cut to 8 cm x 6 cm and attached to a bottomless 24-well microplate (CSC CREA Co., Ltd., product code CSCSM0243). The wells were filled with 70% ethanol and irradiated with UV for 1 hour to check for leakage and to sterilize and disinfect. The 70% ethanol was removed and the plates were washed twice with sterile distilled water. Subsequently, for cell non-adhesion treatment, 1 mL / well of filter-sterilized 1% (w / v) Pluronic F-127 was added, and the Pluronic F-127 solution was filled into the microwells by removing air with a vacuum, and coating was performed at room temperature for 1 hour. After coating, the plates were washed twice with sterile distilled water and replaced with the appropriate culture medium for each cell.

[0035] [Cell Preparation] Human iPS cells (TkDN4-M) were pre-cultured in 6-well tissue culture plates (AGC Technoglass Co., Ltd., product code 3810-006). Vitronectin (VTN-N) Recombinant Human Protein (Thermo Fisher Scientific Co., Ltd., product number A14700), adjusted to 5 μg / mL, was added at 1 mL / well and coated at room temperature for 1 hour. The coating solution was removed immediately before use. Frozen stocks of iPS cells were rapidly thawed in a 37°C water bath, transferred to 5 mL of culture medium, centrifuged (180G, 3 min), and resuspended in medium. The cells were then placed in VTN-N coated plates in a 3.5 x 10⁶ arrangement. 4 cells / cm 2 Cells were seeded in 500 mL / well of culture medium. The culture medium used was Essential-8 (Thermo Fisher Scientific Co., Ltd., product number A1517001) to which the ROCK inhibitor Y-27632 (Fujifilm Wako Pure Chemical Corporation, product code 030-24021) was added to a final concentration of 10 μM. The cells were cultured overnight in a 37°C, 5% CO2 incubator, and the medium was changed to Essential-8 24 hours after seeding.

[0036] Medium specific for iPS cells As a medium specific for iPS cells, Essential 8® Medium (Thermo Fisher Scientific K.K., product No. A1517001) was used.

[0037] Pre-culture of hepatoblastoma-derived cell line HepG2 cells A frozen stock of HepG2 cells was rapidly thawed in a 37°C water bath, transferred into 10 mL of medium, centrifuged (180G, 3 minutes), the supernatant was removed, and the cells were resuspended in medium. The cells were seeded at 1.8 x 10 4 cells / cm 2 onto a 100 mm tissue culture dish (AGC Techno Glass Co., Ltd., product code 3020-100), and cultured for 2 days in a 37°C, 5% CO2 incubator. The medium used was D-MEM (Low Glucose) (FUJIFILM Wako Pure Chemical Corporation, product code 041-29775) supplemented with fetal bovine serum (Biowest, cat. S1810-500) to a final concentration of 10%.

[0038] Medium specific for HepG2 cells As a medium specific for HepG2 cells, a medium prepared by adding fetal bovine serum (Biowest, cat. S1810-500) to D-MEM (Low Glucose) (FUJIFILM Wako Pure Chemical Corporation, product code 041-29775) to a final concentration of 10% was used.

[0039] [Preparation of perfusion culture plate] Prepared PDMS microwell sheets having honeycomb-shaped wells with an inscribed circle diameter (126 µm) and depth aspect ratios of 1:1 and 1:1.5, respectively, were cut to the plate size of a bottomless on-chip pump-type multi-organ MPS device (Sumitomo Bakelite Co., Ltd., product name Biostera™), and after immersing the sheets in 70% ethanol, 70% ethanol was allowed to penetrate into the microwells by suction, followed by standing still for 1 hour, thereby performing liquid leakage confirmation, sterilization and disinfection. This sterilized microwell sheet was dried in a clean bench, and attached to Biostera TM tape) using double-sided tape (manufactured by Teraoka Seisakusho Co., Ltd., Kapton TM ).

[0040] Note that BiosteraTM As shown in FIG. 13, the plate has a structure in which well A and well B are annularly connected via channel 30, channel 30' and channel 50. The medium placed in well A flows from channel 30 to well B through channel 30' driven by a stirrer 40, and the medium in well B flows from channel 50 back to well A.

[0041] Biostera TM comprises 6 sets of the unit. To unify cell seeding and perfusion conditions, 3 units each (N=3) of microwell sheets with an aspect ratio of 1:1 and 1:1.5 are respectively arranged on one Biostera TM plate, and 2 such plates are prepared.

[0042] Biostera with attached microwells TM 70% ethanol was added to all wells of the plate at 1 mL per well, followed by vacuuming for 10 minutes for sterilization. The 70% ethanol was removed with an aspirator, and ethanol was further removed by adding 1 mL of sterile water per well and then removing the sterile water. Next, 1% (w / v) Pluronic F-127 coating solution was added to the Biostera TM According to the plate protocol, 1000 μL of the solution was added to each well B, and then 200 μL was added to each well A. The channels were also filled with 1% (w / v) Pluronic F-127 solution. After about 1 minute, it was confirmed that the coating solution in well B had moved to well A by hydrostatic pressure and the liquid levels became equal. Furthermore, vacuuming was performed to fill the microwells with 1% (w / v) Pluronic F-127 coating solution.

[0043] Next, the plates were UV-irradiated in a clean bench for 1 hour to sterilize and coat with Pluronic F-127 simultaneously. The coating solution was removed with an aspirator, 1 mL of PBS was added to well B, and the plate was allowed to stand for about 1 minute until the PBS flowed into well A and the liquid levels in both wells were approximately the same. Then, the solution was removed from well A with an aspirator. This was repeated three times to remove excess coating solution. Finally, the PBS was removed, and 1.2 mL / well of HepG2 medium was added to well B. After confirming that the liquid levels in wells A and B were approximately the same, the plates were placed in a stirrer dock in a 37°C CO2 incubator for bio-sterilization. TM The plate was set up and perfused overnight at the maximum rotation speed (5000 rpm).

[0044] HepG2 Cell Preparation: Human hepatoblastoma-derived HepG2 cells (cell number: JCRB1054) were purchased from the JCRB Cell Bank of the National Institute of Biomedical Innovation, Health and Nutrition, and then induced dormancy and cultured. The culture medium used was D-MEM (Low Glucose) with L-Glutamine and Phenol Red (Fujifilm Wako Pure Chemical Industries, Ltd., product code: 041-29775) with 10% inactivated fetal bovine serum added.

[0045] As a pre-culture, 3 x 10⁶ HepG2 cells were used. 4 cells / cm 2 Seeds were sown onto four 100mm Tissue Culture Dishes and cultured in a 37°C CO2 incubator for 3 days. The culture medium was removed from the culture dishes using an aspirator, and each was rinsed twice with 10 mL of 0.5 mM EDTA / PBS, and then 3 mL of TrypLE was added. TM Cells were detached after adding Express Enzyme (Thermo Fisher Scientific Inc., product number: 12605028) and treating at 37°C for 6 minutes. 7 mL of specialized culture medium was added to each detached cell, and the entire volume was collected. The cells were then centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. The cell aggregates were resuspended in 10 mL of specialized culture medium, strained through a cell strainer (mesh size: 40 μm), and then the cells were counted.

[0046] 8 x 10 5 When a cell suspension of cells / mL is prepared and seeded into well A at 500 mL / well, the cell concentration is 2 x 10⁶. 5 cells / cm 2 I adjusted it so that it would work.

[0047] [Example 1] Seeding of human iPS cells (TkDN4-M) into PDMS microwell plates The culture medium was removed from the iPS cell culture in a 6-well plate, and PBS was added at 2 mL / well and rinsed off. 5 mM EDTA / PBS was then added at 1 mL / well, incubated at 37°C in a 5% CO2 incubator for 2 minutes, and then removed. Subsequently, 1 mL / well of Essential-8 medium containing 10 μM Y-27632 was added, the cells were collected by pipetting, centrifuged (180 G, 3 min), and the supernatant was removed.

[0048] The harvested cells were suspended in Essential-8 medium containing 10 μM Y-27632, the number of cells was counted, and 6 x 10 were placed in a 24-well PDMS microwell plate with an aspect ratio of 1:1.5. 4 cells / cm 2 The cells were seeded in this manner, and culture was started in a 37°C 5% CO2 incubator. For two days starting the day after cell seeding, 350 mL of culture medium was collected using a micropipette and 450 mL of fresh Essential-8 medium was added to perform a medium change. Before and after this procedure, the state of the cells in the microwells was recorded using a microscope and observed visually. In addition, changes occurring in the same well were recorded during cell culture using a cell monitoring system (CM30 Incubation Monitoring System, Evident Co., Ltd.). The results are shown in Figures 5a1 to 5a4.

[0049] [Comparative Example 1] Cells were cultured in the same manner as in Example 1, except that a 24-well PDMS microwell plate with an aspect ratio of 1:1.5 was used instead of a 24-well PDMS microwell plate with an aspect ratio of 1:1. The cells were then observed using a microscope. The results are shown in Figures 5b1 to 5b4.

[0050] On day 1 after cell seeding, as can be seen in Figures 5a1 and 5b1, relatively uniform-sized spheroids were formed in all microwells, regardless of the aspect ratio. After the culture medium change on day 1, almost no spheroid movement was observed in the wells with an aspect ratio of 1:1.5 (Figure 5a2). In contrast, in the wells with an aspect ratio of 1:1, the spheroids had moved out of the microwells, leaving the wells empty (Figure 5b2). Furthermore, some of the spheroids that had moved out of the wells had moved into other wells, resulting in a state where multiple spheroids were present in a single well.

[0051] On day 2 after cell seeding, homogeneous spheroids are formed in wells with an aspect ratio of 1:1.5 (Figure 5a3). In contrast, in wells with an aspect ratio of 1:1, after changing the culture medium on day 1, microwells containing multiple spheroids show that the spheroids have merged to form a single larger spheroid, resulting in non-uniform spheroid size across microwells (Figure 5b3).

[0052] Figures 5a4 and 5b4 show the results of changing the culture medium on the second day of culture. In the wells with an aspect ratio of 1:1.5, it can be seen that uniform spheroids are formed (Figure 5a4). In contrast, in the wells with an aspect ratio of 1:1, some spheroids move to other wells each time the culture medium is changed, either leaving the well empty or other spheroids fuse together to form a large spheroid, indicating that the productivity of spheroid numbers and size non-uniformity is further exacerbated (Figure 5b4). From these results, it can be inferred that the microwell plate used in Example 1 allows for the formation of spheroids with a uniform shape because cells do not escape from each well even when the culture medium is changed. Furthermore, it can be inferred that stable drug efficacy tests can be performed when performing perfusion culture such as MPS because the cells in each well do not move out of the well due to perfusion.

[0053] [Example 2] Seeding of hepatoblastoma-derived cell line HepG2 cells onto PDMS microwell sheets. The culture medium was removed from the HepG2 cell culture in a 100 mm tissue culture dish, 10 mL of PBS was added, rinsed and removed, and this was repeated twice. Furthermore, 3 mL of TrypLE was added. TM Express (Thermo Fisher Scientific Co., Ltd., product number 12604-021) was added, incubated at 37°C in a 5% CO2 incubator for 5 minutes, and then 7 mL of culture medium was added and the entire mixture was collected.

[0054] The harvested cells were centrifuged (180G, 3 minutes), the supernatant was removed, and the cells were resuspended in culture medium and passed through a 40 mm cell strainer to collect single cells. The number of cells was counted and 6 x 10⁶ were placed in a pre-prepared 24-well PDMS microwell plate. 4 / cm 2 Seeds were seeded to a medium volume of 500 mL / well, and cultivation was started in a 37°C 5% CO2 incubator.

[0055] For two days starting the day after cell seeding, 350 mL of culture medium was collected using a micropipette and 450 mL of fresh medium was added to perform a medium exchange. Before and after this procedure, images of the cells in the microwells were recorded using a microscope, and their condition was observed visually. Furthermore, five days after cell seeding, spheroids were collected by pipetting using a micropipette, confirming that spheroids could be easily collected even from microwells with an aspect ratio of 1:1.5.

[0056] During cell culture, changes occurring in the same well were recorded using a cell monitoring system (CM30 Incubation Monitoring System, Evident Co., Ltd.). The results are shown in Figures 6c1-c5 and Figure 7.

[0057] [Comparative Example 2] Cell culture and its state were observed using a microscope in the same manner as in Example 1, except that a 24-well PDMS microwell plate with an aspect ratio of 1:1 was used instead of a 24-well PDMS microwell plate with an aspect ratio of 1:1.5. The results are shown in Figures 6d1 to d5.

[0058] On day 1 after cell seeding, as can be seen in Figures 6c1 and d1, spheroids of relatively uniform size were formed in all microwells, regardless of the aspect ratio. After the culture medium change on day 1, almost no spheroid migration was observed in the wells with an aspect ratio of 1:1.5 (Figure 6c2).

[0059] In contrast, in wells with an aspect ratio of 1:1, it can be seen that the spheroids have moved out of the microwells, leaving the wells empty (Figure 6d2). Furthermore, some of the spheroids that moved out of the wells have moved into other wells, resulting in a state where multiple spheroids are present in a single well.

[0060] On the second day, homogeneous spheroids were observed to have formed in the wells with an aspect ratio of 1:1.5 (Figure 6c3). In contrast, in the wells with an aspect ratio of 1:1, due to the medium change on the first day of culture, the microwells containing multiple spheroids showed that the spheroids had merged to form a single, larger spheroid, resulting in non-uniform spheroid size across the microwells (Figure 6d3).

[0061] Figures 6c4 and 6d4 show the results of changing the culture medium on the second day of incubation. Figure 6c4 shows that uniform spheroids have been formed. In contrast, in the wells with an aspect ratio of 1:1, some spheroids move to other wells with each culture medium change, either leaving the well empty or causing other spheroids to fuse and form a large spheroid, resulting in further unevenness in spheroid productivity and size (Figure 6d4).

[0062] These results suggest that, using the microwell plate in Example 2, cells do not escape from each well even when the culture medium is changed, making it possible to form spheroids with a homogeneous shape. Furthermore, it can be inferred that when performing perfusion culture such as MPS, the cells in each well do not move out of the well due to perfusion, making it possible to perform stable drug efficacy tests and other similar experiments.

[0063] Furthermore, as can be seen from Figure 7, the spheroids cultured in this microwell plate exhibit very uniform morphology, which is expected to suppress performance variations dependent on spheroid size during MPS and tissue formation.

[0064] [Example 3] Seeding of Hepatoblastoma-derived HepG2 cells onto a PDMS microwell sheet The prepared hepatoblastoma-derived HepG2 cells were placed in a BioStellar well. 400 μL of culture medium was removed from well B, and the sheet was left to stand for about 1 minute to confirm that the liquid levels in wells A and B were approximately the same. 500 μL of the pre-prepared cell suspension was added to well A and 500 μL of the special culture medium was added to well B simultaneously. The cells were seeded to ensure that the liquid levels were equal and no cells entered the flow path. Culture was then started in a 37°C CO2 incubator.

[0065] One hour after sowing, perfusion culture (stirrer speed 4500 rpm) was started on the first plate. For the second plate, static culture was allowed to continue until the next day, and perfusion culture (stirrer speed 4500 rpm) was started 24 hours after sowing.

[0066] For perfusion cultures started one hour after seeding and those started 24 hours after seeding, images of the cells in the microwells were recorded using a microscope at the start of perfusion culture, 24 hours, 48 ​​hours, 72 hours, and 144 hours after seeding, and the condition was observed visually. The results are shown in Figures 8 to 12. Figure a shows the results when perfusion culture was started one hour after seeding, and figure b shows the results when it was started 24 hours after seeding.

[0067] [Comparative Example 3] Cell culture and its state were observed under a microscope in the same manner as in Example 3, except that a biostellar with microwells having an aspect ratio of 1:1 was used instead of a biostellar with microwells having an aspect ratio of 1:1.5. The results are shown in Figures 8 to 12.

[0068] From the results of Example 3 and Comparative Example 3, it can be seen that in Comparative Example 3, regardless of whether the perfusion start time was 1 hour or 24 hours after cell seeding, empty wells were observed after 24 hours of perfusion culture, with cells jumping out of the microwells and moving to other wells. In contrast, in Example 3, all cells remained in the wells and formed cell aggregates (spheroids). This trend became more pronounced over time, with an increase in cells proliferating outside the wells after 48 and 72 hours, and after 144 hours, cells had proliferated outside the wells to such an extent that the wells could no longer be observed. In contrast, in Example 3, all cells were present in the wells at all time points and formed uniform cell aggregates (spheroids).

[0069] Furthermore, regarding the difference between wells where perfusion was started at 1 hour and those where it was started after 24 hours, in Comparative Example 3, it appears that slightly less cell proliferation occurred outside the well when perfusion culture was started after 24 hours. However, there were many microwells where cell aggregates were not formed, and as a result, a uniform and large quantity of cells could not be obtained. Therefore, it is presumed that it would be difficult to conduct stable drug efficacy tests, etc., when used in MPS, etc.

[0070] In contrast, in Example 3, it can be seen that uniform cell aggregates (spheroids) are formed in each well regardless of the start time of perfusion culture. As a result, in the MPS in Example 3, the number and quality of the cell source are uniform, which prevents the occurrence of heterogeneity in effects and results caused by the number of cells between each well and between each system in drug screening and pharmacokinetic studies. Therefore, it can be inferred that evaluation can be performed in an environment closer to that of a living organism, and that stable drug efficacy tests can be conducted. The microwell sheet of the present invention enables the formation of stable cell aggregates (spheroids), providing a stable cell source useful for drug interaction tests such as MPS, and allowing for the efficient and large-scale acquisition of necessary cells even under perfusion culture.

Claims

1. A microwell sheet having concave microwells having an opening and a flat bottom, wherein the depth from the opening to the bottom is at least 1.4 times the diameter of the opening.

2. The microwell sheet according to claim 1, wherein the shape of the opening and the opening surface and the bottom surface of the opening and bottom are honeycomb-shaped.

3. The microwell sheet according to claim 1, wherein one adjacent microwell has a wall portion of equal thickness between it and another microwell.

4. A microwell sheet according to claim 1, which is gas permeable.

5. The microwell sheet according to claim 1, wherein the thickness of the bottom portion is 0.8 μm or less.

6. The microwell sheet according to claim 1, which is made of silicone resin.

7. A cell culture method comprising the step of forming spheroids or organoids by culturing cells in the microwells of a microwell sheet according to claim 1.

8. A method for producing a spheroid or organoid, comprising the step of forming a spheroid or organoid by culturing cells in the microwells of a microwell sheet according to claim 1.

9. The method according to claim 7, wherein the culture is perfusion culture.

10. The method according to claim 8, wherein the culture is perfusion culture.

11. A microphysiological system comprising a microwell sheet as described in claim 1 and a container for arranging the microwell sheet.

12. A method for evaluating a drug, comprising the steps of culturing a spheroid or organoid placed in a microwell of the microphysiological system described in claim 11 while supplying gas, and introducing the drug to be evaluated into the container of the microphysiological system.

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