Method for manufacturing cell culture sheets, method for manufacturing cell culture food, jig for manufacturing cell culture sheets, and apparatus for manufacturing cell culture sheets.

Abstract

To provide a method for producing cell-based food, which can easily produce three-dimensionally shaped food made of cultured cells with no need of installing a hydrogel scaffold.SOLUTION: Provided is a method for producing a cell culture sheet, Including the steps of: introducing a filling liquid containing spheroids and a dispersion medium into a gap between a pair of plate-shaped liquid-permeable facing members disposed to face each other at an arbitrary interval to fill the gap with the spheroids in a state in which the dispersion medium can freely permeate the liquid-permeable facing members; causing neighboring spheroids to adhere to each other in a state that the spheroids are alive, to thereby form a cell culture sheet; and taking out the cell culture sheet from the gap.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a cell culture sheet and a method for producing the same, a cell culture food and a method for producing the same, a jig for producing a cell culture sheet, and a cell culture sheet production apparatus.

Background Art

[0002] In recent years, with the global population increase, the demand for food has been rising, and there is concern about the shortage of meat as a protein source. On the other hand, there are many problems in terms of land, feed, excrement treatment, labor force securing, etc. for increasing livestock production. As a way to leapfrog these problems, the production of artificial meat by cell culture has been proposed (for example, Patent Documents 1 and 2).

[0003] In Patent Document 1, a hydrogel containing skeletal myoblast cells is formed into a substantially rectangular shape, and a plurality of cell modules having holes of a predetermined shape are prepared in the hydrogel. The plurality of cell modules are stacked so that the shapes of the holes do not overlap when viewed from above, and in this state, the skeletal myoblast cells are proliferated and cultured, and further induced to differentiate into muscle tubes, reporting that a three-dimensional muscle tissue with a texture close to conventional edible meat can be obtained.

[0004] In Patent Document 2, an idea of forming a three-dimensional scaffold made of a hydrogel and seeding a population of self-renewing cells thereon to produce meat suitable for consumption is disclosed.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] The conventional technology described above involves shaping a hydrogel, which serves as a scaffold for cells, into a three-dimensional form, culturing cells within it, and inducing differentiation. Therefore, conventional technology requires the effort of setting up the hydrogel as a scaffold, and there were concerns that the culture period would be prolonged. In addition, there were concerns that the large amount of hydrogel contained in the finished meat sample would impair the flavor and texture of the meat.

[0007] Therefore, one of the objectives of the present invention is to provide a method for producing cell-cultured foods that can be easily manufactured in a three-dimensional shape consisting of cultured cells without the need to install a hydrogel scaffold. [Means for solving the problem]

[0008] The present invention includes the following embodiments. [1] A method for manufacturing a cell culture sheet, comprising the steps of: introducing a filling solution containing spheroids and a dispersion medium into the gap between a pair of plate-shaped liquid-permeable opposing members arranged opposite each other at an arbitrary distance apart, filling the gap with spheroids in such a state that the dispersion medium can freely permeate the liquid-permeable opposing members; forming a cell culture sheet by adhering adjacent spheroids together while the spheroids filling the gap are alive; and removing the cell culture sheet from the gap. [2] A cell culture sheet formed by the adhesion of adjacent spheroids. [3] A method for producing a cell culture food, comprising the steps of obtaining a plurality of cell culture sheets by the manufacturing method described in [1], and stacking the plurality of cell culture sheets to form a thick cell culture food. [4] A cell culture food made of multiple stacked cell culture sheets, each consisting of adjacent spheroids adhering to each other. [5] A jig for manufacturing cell culture sheets, comprising: a pair of plate-shaped liquid-permeable opposing members arranged facing each other at an arbitrary distance apart; a side member surrounding the space formed by the gap between the liquid-permeable opposing members and sealing the space; and an opening provided at least one arbitrary location on the side member for communication between the space and the outside, wherein the liquid-permeable opposing members have a number of through holes, and the opening diameter of the through holes is 500 μm or less. [6] The cell culture sheet manufacturing jig according to [5], wherein at least a portion of the liquid permeable opposing member is formed of a metal or resin mesh member. A cell culture sheet manufacturing apparatus comprising a jig for manufacturing cell culture sheets as described in [7] [5] or [6], and a container capable of accommodating part or all of the jig for manufacturing cell culture sheets. [Effects of the Invention]

[0009] In the method for producing cell culture food of the present invention, first, spheroids, which are aggregates of cells, are filled into a predetermined sheet-like space, and by allowing them to be active in a living state for a while, a cell culture sheet is formed in which the spheroids adhere to each other. Next, by stacking a desired number of cell culture sheets, a cell culture food of a certain thickness can be easily obtained. According to the present invention, the three-dimensional hydrogel scaffold required in conventional techniques is unnecessary, and the long culture period for cell proliferation and differentiation is also unnecessary. Furthermore, the resulting cell culture food does not need to contain hydrogel, so it is essentially a blank slate, and it is easy to adjust the texture and flavor by adding desired components as needed. [Brief explanation of the drawing]

[0010] [Figure 1] A schematic diagram of the method for producing cell culture food according to the present invention. [Figure 2] A schematic perspective view showing an example of a cell culture jig according to the present invention. [Figure 3] (a) A schematic front view showing an example of how to use the cell culture jig according to the present invention. (b) A cross-sectional view taken along the line XX in the front view. [Figure 4] A schematic perspective view showing an example of a cell culture sheet manufacturing apparatus according to the present invention. [Modes for carrying out the invention]

[0011] Embodiments of the present invention will be described below with reference to the drawings. The jigs and devices shown are schematic diagrams illustrating an example of an embodiment, and their actual dimensions and shapes may differ.

[0012] Figure 1 shows an overview of the method for producing the cell culture food of the present invention. First, desired cells are cultured to form spheroids, and then multiple spheroids are arranged in a sheet-like structure and adhered to each other to obtain a cell culture sheet. A desired number of these sheets are then stacked to produce a cell culture food with thickness. The technical elements for obtaining the cell culture food of the present invention will be explained below.

[0013] ≪Spheroid≫ Spheroids are aggregates of cells, usually roughly spherical in shape. Their size (diameter) varies depending on the cell type, but is typically around 0.1 to 0.5 mm. The type of cells that form spheroids is not particularly limited and can be selected from any cells derived from cattle, pigs, or chickens that are conventionally used for food. From the viewpoint of producing large quantities of homogeneous spheroids, immortalized cells that do not have limitations on the number of subculturing cycles are preferred. The cells and spheroids, which are aggregates thereof, used in this invention are obtained by known methods.

[0014] ≪Method for manufacturing cell culture sheets≫ A first aspect of the present invention is a method for producing a cell culture sheet comprising a first to third step. <First step> The first step is to fill a sheet-like space with spheroids. This space can be formed, for example, by facing two plate-like members towards each other at a predetermined separation distance and using the gap between these plate-like members. However, from the perspective of sufficiently supplying the culture solution to the filled spheroids, it is important that the plate-like members have the property of permeating liquid.

[0015] Therefore, in the first step, it is preferable to introduce a filling liquid containing spheroids and a dispersion medium into the gap between a pair of plate-like liquid-permeable opposing members arranged to face each other at an arbitrary interval, and fill the gap with the spheroids in a state where the dispersion medium can freely permeate the liquid-permeable opposing members. Examples of the dispersion medium include known liquid media suitable for the cells constituting the spheroids, such as MEMα, D-MEM, E-MEM, RPMI, etc. Among these, MEMα is preferable from the perspective of improving the culture efficiency of the spheroids. Also, in order to prevent the filled spheroids from spilling out of the gap, it is preferable that the outer periphery of the gap is sealed by an arbitrary member.

[0016] [[ID=J]] When an inlet is opened in a part of the member that seals the outer periphery and the filling liquid is introduced, the dispersion medium introduced together with the spheroids permeates the liquid-permeable opposing members and flows out to the outside of the gap. On the other hand, the spheroids introduced into the gap do not pass through the liquid-permeable opposing members and remain in the gap. As a result, the gap can be filled with spheroids at a high density so as to completely fill the gap.

[0017] (Fixture for manufacturing cell culture sheet) Figure 2 shows an example of a cell culture sheet manufacturing jig suitable for carrying out the first step. The illustrated cell culture sheet manufacturing jig 1 (hereinafter simply referred to as "jig 1") comprises a first plate-shaped mesh member 2 and a second plate-shaped mesh member 3, which are a pair of plate-shaped liquid permeable opposing members made of tungsten or the like and arranged facing each other at an arbitrary distance apart; a first spacer 4, a second spacer 5, a third spacer 6, a fourth spacer 7, and a fifth spacer 9, which are side members that surround the space formed by the gap between these liquid permeable members and seal the space; and an opening 8 provided at least one arbitrary location on the side members that connects the space to the outside.

[0018] The liquid-permeable counter member can have numerous through-holes that do not allow spheroids contained in the filling liquid introduced through the opening 8 to pass through, but allow the dispersion medium contained in the filling liquid to pass through in a short time. Examples include mesh members, plates made of tungsten or the like with numerous through-holes in the thickness direction, nonwoven fabrics, and woven fabrics. If the material of these liquid-permeable counter members is flexible and cannot maintain a plate shape on its own, it can be maintained in a plate shape by, for example, using a frame and stretching the liquid-permeable counter member across the frame.

[0019] The opening diameter of each through-hole in the liquid-permeable opposing member is preferably 500 μm or less, and more preferably 300 μm or less, from the viewpoint of preventing spheroid permeation. Furthermore, from the viewpoint of easily allowing liquid permeation, the opening diameter of the through-holes is preferably 50 μm or more, and more preferably 100 μm or more. The opening diameter of the mesh member is expressed by the pitch of the wires constituting the mesh.

[0020] From the viewpoint of fully exhibiting the above functions and being easy to handle, it is preferable that the portion of the liquid-permeable opposing member that comes into contact with the spheroid is formed of a metal or resin mesh member. Furthermore, a metal mesh member is more preferable because it is easy to sterilize and has excellent durability. Tungsten is preferred as the metal constituting the mesh member because it has high biocompatibility and is easy to mold.

[0021] The spacing (distance between) the plate-shaped mesh members 2 and 3 is preferably 1.0 to 3.0 times the diameter of the spheroid to be filled, more preferably 1.0 to 2.0 times, and even more preferably 1.2 to 1.5 times. With this spacing, when spheroids are filled into the gap (the space sandwiched between the plate-shaped mesh members), a cell culture sheet is formed in the plane direction of the plate-shaped mesh members 2 and 3, and its thickness can be made to be about the thickness of 1 to 3 spheroids. From the viewpoint of forming a cell culture sheet of uniform thickness, it is preferable that the main surfaces of the pair of liquid-permeable opposing members are arranged parallel to each other.

[0022] The jig 1 has a space enclosed by plate-shaped mesh members 2 and 3 and side members 4 to 7 and 9. The space is sealed by each member except for an opening 8 that communicates with the outside. Since spheroids do not permeate the plate-shaped mesh members 2 and 3, the space can be filled with spheroids by introducing a large number of spheroids through the opening 8.

[0023] When the filling liquid is introduced through the opening 8 provided between the fourth spacer 7 and the fifth spacer 9, the filling liquid flows down in the direction of gravity, and the spheroids gradually pile up from the third spacer 6, which supports the bottom of the space, toward the opening 8, until the space is eventually filled to the brim with spheroids.

[0024] In the illustrated example, the first wire α constituting the plate-shaped mesh members 2 and 3 is oriented vertically downwards, and the second wire β is oriented perpendicular (horizontal) to the first wire. However, the orientation of each wire is not limited to this example. The first and second wires may be rotated 90 degrees clockwise relative to the illustrated example and oriented in a direction intersecting the vertical downward direction.

[0025] In the illustrated example, one opening 8 is provided in the ceiling portion formed by the fourth spacer 7 and fifth spacer 9 of jig 1, but the position and number of openings are not limited to this example. For example, two or more openings 8 may be provided in the ceiling portion. In addition, one or more openings with a diameter smaller than a spheroid may be provided in the side portion formed by the first spacer 4 or the second spacer 5.

[0026] In the illustrated example, the filling liquid is introduced from the top to the bottom of the jig 1 along the direction of gravity, but the direction in which the filling liquid is introduced may be set independently of the direction of gravity. For example, the filling liquid may be introduced horizontally from an opening provided on the side, and the spheroid may be dispersed using the force of the jet to fill the space.

[0027] To facilitate the introduction of the filling liquid containing spheroids through the opening 8 of the jig 1, it is preferable to use a funnel connected to the opening 8. An example of such a funnel is the adapter 12 shown in Figure 3. The adapter 12 is a plate-shaped member that covers the top of the jig 1 and has a funnel-shaped spout 12a connected to the opening 8. The spout 12a penetrates the adapter 12 in the thickness direction and widens from the bottom connected to the opening 8 upwards, so that the filling liquid can be easily poured in.

[0028] The frame material 11 in Figure 3 is a plate material with a hole punched out in the center, and the jig 1 is fixed by sandwiching it between two opposing frame materials 11. The frame portion that makes up the frame material 11 follows the outer circumference of the mesh members 2 and 3 of the jig 1, and bolts and nuts are used to fix it. The frame material 11 also serves to increase the area of ​​the installation site for the adapter 12.

[0029] After filling the space in the jig 1 with spheroids, the spacing between the plate-shaped mesh members 2 and 3 may be narrowed to reduce the volume of the space in order to increase the filling rate. One way to achieve this is to make each of the spacers 4 to 7 and 9 out of rubber. By pressing the plate-shaped mesh members 2 and 3 together without crushing the spheroids, the rubber spacers are compressed, and the spacing between them can be narrowed. If the spacing is to be kept constant without narrowing it, each of the spacers 4 to 7 and 9 may be made of metal such as stainless steel or resin such as fluororesin.

[0030] <Second process> The second step is to form a cell culture sheet by causing adjacent spheroids, while they are still alive, to adhere to each other while filling the gap between the pair of plate-shaped liquid-permeable opposing members.

[0031] To keep the filled spheroids alive, it is preferable that each spheroid be in contact with fresh culture medium. Since the liquid-permeable opposing member allows the culture medium to pass through, it is preferable that fresh culture medium naturally diffuses from the outside into the gap. Spheroids in contact with fresh culture medium can continue their life activities, and adjacent spheroids naturally adhere to each other, forming a cell culture sheet.

[0032] Figure 4 shows an example of a jig 1, in which spheroids are filled into the gaps, being immersed in a fresh culture medium M filled in a container 22. The culture medium M permeates through the plate-shaped mesh members 2 and 3 and diffuses freely inside and outside the jig 1, so that all the filled spheroids are always in contact with the fresh culture medium M. If this state is maintained for the number of days necessary for the spheroids to adhere to each other (e.g., 1 to 5 days), the desired cell culture sheet will be formed inside the jig 1.

[0033] The composition of the culture medium M is not particularly limited, and any known composition that preserves the vital activity of the spheroids can be used. Examples include MEMα. Known additives that induce the differentiation of each cell constituting the spheroids may be included in the culture medium M. The temperature of the culture medium M is preferably within a temperature range that preserves the vital activity of the spheroids. The pH, CO2 concentration, oxygen concentration, temperature, etc. of the culture medium M can be controlled by general culture methods.

[0034] In addition, in the first step described above, an empty jig 1, which has not yet been filled with spheroids, may be immersed in the culture medium M as shown in Figure 4, and a filling solution containing spheroids may be introduced into the jig 1 through the opening 8 via a nozzle or adapter 12 (not shown), thereby filling the gap with spheroids. With this method, the spheroids can be gently filled while always in contact with the culture medium M, thus reducing stress on the spheroids and allowing for rapid adhesion between spheroids in the second step.

[0035] <Third step> The third step is to remove the cell culture sheet from the gap. If jig 1 is used in the first and second steps, the jig 1 is dismantled in the third step, and the cell culture sheet is left on one plate-shaped mesh member 2 while the other plate-shaped mesh member 3 is removed to obtain the desired cell culture sheet.

[0036] Cell culture sheet A second aspect of the present invention is a cell culture sheet formed by the adhesion of adjacent spheroids. The cell culture sheet of this embodiment can be obtained by the manufacturing method of the first embodiment. The use of the cell culture sheet according to this embodiment is not particularly limited, and it may be used as a material for cell culture foods described later, as well as as a material for pharmaceuticals or as a test material for pharmaceutical development.

[0037] The cell culture sheet may contain one type of spheroid, or two or more types. The individual spheroids contained in the cell culture sheet may consist of one type of cell, or two or more types of cells. There is no particular limit to the number of cells that make up each spheroid contained in a cell culture sheet; it depends on the size of the spheroid, but it can range from several hundred to several thousand.

[0038] In a cell culture sheet, individual spheroids are adjacent to and adhere to each other along the planar direction of the sheet. The preferred form of adhesion is one in which cells present on the spheroid surface spontaneously adhere to each other, for example, by binding to each other via proteins such as fibronectin present on the cell surface.

[0039] Ideally, the thickness of the cell culture sheet should be the thickness of a single spheroid, without the spheroids stacking in the thickness direction. For example, if the diameter of the spheroids constituting the cell culture sheet is 0.1 to 0.5 mm, the thickness of the cell culture sheet should preferably be 0.1 to 1.0 mm. The thickness of the cell culture sheet is determined as the average value of the thicknesses of 10 arbitrarily selected cross-sections measured using a known method such as a measuring microscope. The length and width of the cell culture sheet are not particularly limited and can be arbitrarily adjusted within a range of 10 to 100 cm in both length and width, depending on the size of the jig used to manufacture the cell culture sheet.

[0040] Cell culture sheet manufacturing equipment A third aspect of the present invention is a cell culture sheet manufacturing apparatus comprising a jig for manufacturing cell culture sheets and a container capable of housing part or all of the jig for manufacturing cell culture sheets.

[0041] The cell culture sheet manufacturing jig comprises a pair of plate-shaped liquid-permeable opposing members arranged facing each other at an arbitrary distance apart, a side member surrounding the space formed by the gap between the liquid-permeable opposing members and sealing the space, and an opening provided at least one arbitrary location on the side member that connects the space to the outside. Specifically, examples include the cell culture sheet manufacturing jig 1 described in the first embodiment, and a frame material 11 and adapter 12 that may be provided as needed.

[0042] The container is not particularly limited as long as it has a volume capable of accommodating part or all of the jig for manufacturing the cell culture sheet. By housing the jig for manufacturing the cell culture sheet in this container and also filling it with culture medium, part or all of the jig for manufacturing the cell culture sheet can be immersed in the culture medium. If this apparatus is used in the first or second step of the first embodiment, the manufacturing of the cell culture sheet will be facilitated. The schematic configuration of this embodiment is illustrated in the cell culture sheet manufacturing apparatus 21 in Figure 4.

[0043] ≪Method for producing cell cultured foods≫ A fourth aspect of the present invention includes the steps of obtaining a plurality of cell culture sheets by the manufacturing method of the first aspect, and stacking the plurality of cell culture sheets to form a thick cell culture food. This is a method for producing cell culture foods.

[0044] The method for stacking the cell culture sheets is not particularly limited; they may be stacked sequentially in clean air, or they may be stacked sequentially while immersed in an isotonic solution such as culture medium or physiological saline.

[0045] There are no particular restrictions on the number of cell culture sheets to be stacked; for example, 2 to 1000 sheets are possible. If each cell culture sheet is 0.5 mm thick, stacking 100 sheets results in a thickness of 5 cm. 5 cm is a thickness that is easily handled as ordinary food, such as meat.

[0046] It is difficult to supply fresh culture medium to spheroids inside the layered cell culture food. Therefore, the adhesion between individual cell culture sheets constituting the cell culture food does not need to be relied upon by the life activities of the spheroids, but rather should be achieved by the weight of the cell culture sheets and the physicochemical adhesion of their surfaces.

[0047] ≪Cell culture food≫ A fifth aspect of the present invention is a cell culture food comprising multiple stacked cell culture sheets, each sheet being formed by the adhesion of adjacent spheroids to one another. The cell culture food according to this embodiment can be obtained by the manufacturing method according to the fourth embodiment. In this embodiment of cell culture food, desired components and materials that are added to conventional foods may be added as needed to adjust the texture and flavor. [Examples]

[0048] After assembling the cell culture sheet manufacturing jig 1, which is schematically illustrated in Figures 2-3, the entire cell culture sheet manufacturing jig was sterilized by autoclaving. The dimensions of the plate-shaped mesh members 2 and 3 incorporated into the cell culture sheet manufacturing jig 1, including their outer frames, are 47 mm x 47 mm, and the spacing between opposing mesh members 2 and 3 is 0.7 mm. Mesh members 2 and 3 are made of PTFE (fluoropolymer) mesh (opening diameter: 150 μm), while other components such as the frame are made of SUS (stainless steel).

[0049] After sterilization, the cell culture sheet manufacturing jig 1 was moved into a clean bench. Inside the clean bench, a filling solution was manually introduced through the opening 8 of the cell culture sheet manufacturing jig 1 using a pipette. This solution consisted of approximately 2000 spheroids (clumps of mouse-derived fibroblast-like adherent cells, cell name: MC3T3-E1, size: approximately Φ0.5 mm) and 160 ml of prepared MEMα (ThermoFisher, model number: C12571500BT, FBS: 10%, antibiotic PS: 1%), which is used as a dispersion medium. Subsequently, the opening 8 was covered with an adapter, the cell culture sheet manufacturing jig 1 was placed inside container 22, and 160 ml of new prepared MEMα was introduced into it. The container 22 was set to allow MEMα to permeate the mesh members 2 and 3, and then placed in a CO2 incubator (manufactured by PHC Corporation, model number: MCO-170AICUVD-PJ) adjusted to an environment of 37°C and a CO2 concentration of 5% for 3 days.

[0050] After standing for three days, the cell culture sheet manufacturing jig 1 was removed from the CO2 incubator and moved into a clean bench. The cell culture sheet manufacturing jig 1 was dismantled, and the other plate-shaped mesh member 3 was manually removed, leaving the target cell culture sheet on one plate-shaped mesh member 2. A cell culture sheet (14 mm x 14 mm) was obtained using the above method.

[0051] The obtained cell culture sheets were observed using a fluorescence microscope with CFSE reagent (Dojin Chemical Laboratories, model number: C375), confirming that the cells constituting the cell culture sheets were alive. By observing how the spheroids in the material bound together, it was possible to confirm that the desired cell culture sheets had been produced. [Explanation of symbols]

[0052] 1. Fixture for manufacturing cell culture sheets 2. First plate-shaped mesh member 3. Second plate-shaped mesh member 4. First spacer 5. Second spacer 6. Third spacer 7. Fourth Spacer 8 openings 9. Fifth Spacer 11 Frame material 12 adapters 21 Cell culture sheet manufacturing apparatus 22 Container

Claims

1. A pair of plate-shaped, liquid-permeable opposing members that are inaccessible to spheroids and are arranged facing each other at an arbitrary distance apart, A side member surrounds the space formed by the gap between the liquid-permeable opposing members and seals the space so that the spheroid introduced into the space does not spill out, Using a jig that includes an opening provided at least one arbitrary location on the side member, which connects the space with the outside, A step of introducing a filling liquid containing a spheroid and a dispersion medium into the space formed by the gap between the liquid-permeable opposing members through the opening, and filling the gap with the spheroid in a state in which the dispersion medium can freely permeate the liquid-permeable opposing members and the spheroid does not permeate the liquid-permeable opposing members, The process involves forming a cell culture sheet by allowing adjacent spheroids, while they are still alive, to adhere to each other while filling the gaps. The process includes removing the cell culture sheet from the gap, A method for manufacturing cell culture sheets.

2. The method for manufacturing a cell culture sheet according to Claim 1, wherein the filling liquid flows down from the opening in the direction of gravity, and the spheroids are gradually piled up from the portion of the side member that supports the bottom of the space toward the opening.

3. A method for manufacturing a cell culture sheet according to claim 1, wherein after filling the space with the spheroid, the distance between the pair of plate-shaped liquid-permeable opposing members is narrowed to reduce the volume of the space.

4. A step of obtaining a plurality of cell culture sheets by the manufacturing method described in claim 1, A method for producing a cell culture food, comprising the step of stacking multiple cell culture sheets to form a thick cell culture food.

5. A jig for manufacturing a cell culture sheet, to be used as the jig in the manufacturing method described in Claim 1, A pair of plate-shaped, liquid-permeable opposing members that are not permeable to spheroids, arranged facing each other at an arbitrary distance apart, A side member surrounds the space formed by the gap between the liquid-permeable opposing members and seals the space so that the spheroid introduced into the space does not spill out, The side member comprises an opening provided at least one arbitrary location that connects the space to the outside, A jig for manufacturing cell culture sheets, wherein the liquid-permeable opposing member has numerous through holes, and the opening diameter of the through holes is 500 μm or less.

6. The cell culture sheet manufacturing jig according to claim 5, wherein at least a portion of the liquid-permeable opposing member is formed of a metal or resin mesh member.

7. A cell culture sheet manufacturing apparatus for use in the manufacturing method described in Claim 1, The jig for manufacturing the cell culture sheet according to claim 5 or 6, A container capable of housing part or all of the jig for manufacturing the cell culture sheet, A cell culture sheet manufacturing apparatus equipped with the following features.

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