JP6384054B2 - Cell culture container for producing cell laminate - Google Patents

Description

  The present invention relates to a cell culture vessel for producing a cell laminate in which a plurality of cells are laminated in the thickness direction, and a method for producing a cell laminate using the cell culture vessel.

  A biological tissue produced by culturing cells on a culture carrier such as collagen is not suitable as a tissue for transplantation for medical purposes because of its low cell density. A technique for producing a tissue such as a cell sheet having a high cell density suitable for transplantation is an important technique in the field of tissue engineering. However, there are several problems in the conventional method for producing a high cell density biological tissue.

  It is generally widely practiced to seed cells on a culture support to form a cell sheet. For the purpose of facilitating cell sheet peeling, a technology that promotes cell peeling by providing a layer of temperature-responsive polymer compound on the cell adhesion surface has been developed, and it is possible to collect cell sheets containing almost no foreign matter. It is. However, the cell sheet formed by this method is usually composed of a single layer or three or less cell layers. For this reason, in order to carry out multilayering, it is necessary to superimpose a plurality of cell sheets. Since cell sheets are extremely thin and difficult to handle, it is not easy to superimpose a plurality of cell sheets, which is troublesome. For example, in Non-Patent Document 1, it took 3 weeks for a chondrocyte collected from a rabbit to be formed into a sheet using a culture dish having a temperature-responsive polymer layer, and to be laminated in a three-layered state that can be transplanted. There is a description. Accordingly, there has been a demand for the development of a method that can easily produce a multilayer cell stack.

  Patent Documents 1 to 3 describe an attempt to form a cell stack by using a load such as centrifugal force and to provide for transplantation. These methods are characterized in that a multi-layered cell stack can be produced at a time. Furthermore, Patent Documents 2 and 3 employ a method of increasing cell-cell adhesion by further culturing after producing a cell laminate by centrifugation.

JP 2010-161954 JP 2010-226962 A JP 2010-46053 A Kaneshiro et al., Biochemical and Biophysical Research Communications 349 723-731 2006

  In the prior art, the cell stack was transferred to another cell culture vessel for further culturing after centrifugation, but at that time, it was found that there was a problem that the cell stack was broken. . In addition, if the cell stack is continued without being transferred to another cell culture vessel, the amount of medium relative to the number of cells will be insufficient, and the cells in the cell stack will die during the culture. It turned out to be seen.

  An object of the present invention is to provide a means for obtaining a cell laminate having high cell-cell adhesion and high strength.

  The present inventors divided the inside of the culture vessel into a plurality of accommodating portions by detachable partition walls, formed cell stacks by centrifugal force in some of the accommodating portions, removed the partition walls, and accommodated wider By further culturing the cell laminate in the part, it was found that the cell-cell adhesion of the cell laminate can be enhanced, and the present invention was completed.

That is, the present invention includes the following inventions.
(1) A cell culture container for producing a cell laminate,
A cell non-adherent inner bottom surface, and a first partition wall and a second partition wall disposed on the inner bottom surface,
The inner bottom surface and the first partition form a first accommodating part capable of accommodating cells and a medium,
The inner bottom surface, the first partition wall, and the second partition wall form a second storage portion that can store cells and a culture medium,
The cell culture container, wherein the first partition wall is detachably attached to the inner bottom surface.
(2) The cell culture container according to (1), wherein the first partition and the second partition are cylindrical, and the first partition is disposed inside the second partition.
(3) The cell culture container according to (1) or (2), wherein the first partition is formed of an elastomer material.
(4) A method for producing a cell laminate,
a) preparing a cell culture container,
The cell culture container includes a cell non-adhesive inner bottom surface, and a first partition wall and a second partition wall disposed on the inner bottom surface,
The inner bottom surface and the first partition form a first accommodating part capable of accommodating cells and a medium,
The inner bottom surface, the first partition wall, and the second partition wall form a second storage portion that can store cells and a culture medium,
The first partition is detachably attached to the inner bottom surface, and b) the step of adding cells and culture medium to the first accommodating portion;
c) performing cell culture while applying centrifugal force toward the inner bottom surface to form a cell stack,
d) adding a culture medium to the second container;
e) The production method comprising a step of removing the first partition, and f) a step of culturing the cell laminate.
(5) The method according to (4), wherein the first partition and the second partition are cylindrical, and the first partition is disposed inside the second partition.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to manufacture a cell laminated body with strong intercellular adhesive force and high intensity | strength.

FIG. 1 is a schematic view showing one embodiment of the cell culture container of the present invention. A1 and B1 are top views, and A2 and B2 are cross-sectional views. A1 and A2 show a state where the first partition is arranged, and B1 and B2 show a state where the first partition is removed. FIG. 2 is a schematic view showing one embodiment of the method for producing a cell laminate of the present invention. FIG. 3 is a photograph showing one embodiment of the cell culture container of the present invention. FIG. 4 is a photograph showing the cell stack. FIG. 5 is a table summarizing the operations in Test Example 1. FIG. 6 is a table summarizing the collection results of the cell laminates in the test examples. FIG. 7 is a photograph showing the layer structure of the cell stack.

  The cell culture container of the present invention is a cell culture container for producing a cell laminate, and includes a cell non-adhesive inner bottom surface, and first and second partition walls arranged on the inner bottom surface. The inner bottom surface and the first partition wall form a first storage portion that can store cells and a medium, and the inner bottom surface, the first partition wall, and the second partition wall can store cells and a medium. Forming a housing portion. At least the first partition wall is detachably attached to the inner bottom surface. The cell culture container of the present invention is preferably a cell culture container for producing a cell laminate by applying a centrifugal force toward the inner bottom surface.

  The cell culture container of the present invention has at least two partition walls and at least two storage portions, but may have further partition walls and further storage portions.

  In one embodiment, the 1st partition and the 2nd partition are cylindrical, and the 1st partition is arranged inside the 2nd partition. As one aspect of such an embodiment, for example, the cell culture container shown in FIG. 1 can be exemplified. A cell culture container 1 shown in FIG. 1 has a structure in which a cylindrical first partition 4 is arranged on the inner bottom surface of a culture dish having an inner bottom surface 2 and a side wall 3. In such a structure, the side wall 3 of the culture dish constitutes the second partition wall. The inner bottom surface 2 and the first partition 4 form a first housing portion 5, and the inner bottom surface 2, the first partition 4, and the second partition 3 (the side wall of the culture dish) serve as a second housing. Part 6 is formed. By using a culture dish having an inner bottom surface and a side wall, the cell culture container of the present invention can be easily produced simply by disposing the first partition wall on the inner bottom surface.

  The culture dish is not particularly limited, but the opening is preferably circular, the opening width (for example, R in FIG. 1) is preferably 25 to 60 mm, particularly 35 mm, and the depth (for example, L1 in FIG. 1). Is preferably 10 to 25 mm. This is the same size as a petri dish used for conventional cell culture, and can be easily produced from a general-purpose petri dish, and is easily adapted to existing culture devices, and therefore, the one having the above size is preferable. .

  The material constituting the inner bottom surface and the second partition, in one embodiment, the culture dish is not particularly limited. Specifically, inorganic materials such as metals, glass, ceramics, silicon, elastomers, plastics (for example, polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluorine resin, polycarbonate resin, polyurethane) Resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin).

  The first partition wall is preferably cylindrical, and the cross-sectional shape perpendicular to the axis of the cylinder is not particularly limited. For example, a circular shape, an elliptical shape, and a regular polygon (regular triangle, square, rhombus, regular pentagon, Regular hexagon, regular octagon, etc.), preferably circular. By using a cylindrical partition wall having a circular or regular polygonal cross section as the first partition wall, it can be stably placed on the inner bottom surface of the container, and centrifugal force is applied in the direction of the inner bottom surface of the cell culture container. At the same time, it is possible to prevent the first partition from moving and thereby the medium and cells stored in the first storage portion from leaking out. More preferably, a cross-sectional shape perpendicular to the axis of the cylinder is point symmetric. By using a cylindrical partition wall whose cross-sectional shape is point-symmetric, a cell laminate having a uniform thickness in the plane can be produced. By making a point-symmetric figure, for example, when swinging with a centrifuge, it is possible to prevent unevenness in the number of cells up and down and the center of gravity to deviate from the center and become non-uniform.

  The cylindrical first partition wall is preferably arranged so that the center of gravity of the figure formed by a cross section perpendicular to the axis thereof overlaps the center of gravity of the inner bottom surface. For example, when the inner bottom surface is circular and the shape formed by the cross section of the first partition is circular, it is preferable to arrange the first partition so that the centers of both circles overlap (FIG. 1). . Thereby, it can be stably arranged on the inner bottom surface of the container, and when the centrifugal force is applied in the direction of the inner bottom surface of the cell culture container, the first partition wall moves and thereby the first accommodating portion It is possible to prevent the contained medium and cells from leaking out.

  The size of the first partition is not particularly limited as long as it is a size that can be accommodated inside the second partition, and in one embodiment, can be accommodated inside the side wall of the culture dish. For example, when the second partition is cylindrical, preferably the opening is a side wall of a circular culture dish, and the first partition is cylindrical, a cross section perpendicular to the axis of the first partition is formed. The diameter of the outer circumference of the circle (for example, r1 in FIG. 1) is preferably smaller than the diameter of the inner circumference of the circle formed by the second partition wall (for example, R in FIG. 1). For example, when a dish having a size of 35 mm is used as the second partition (R = 35 mm), the diameter of the outer periphery of the circle formed by the cross section perpendicular to the axis of the first partition (for example, r1 in FIG. 1) is preferably 6 The inner diameter (for example, r2 in FIG. 1) is preferably 5 to 25 mm.

  The thickness of the first partition can be appropriately designed depending on the material and is not particularly limited, but is preferably 1 to 10 mm. By setting the thickness to a certain value or more, it is possible to withstand the water pressure applied from the culture medium when a centrifugal force is applied. By setting the thickness to a certain value or less, waste of space can be eliminated.

  The height of the first partition (for example, L2 in FIG. 1) is not particularly limited, but the height of the second partition, in one embodiment, the height of the side wall of the culture dish (for example, L1 in FIG. 1). Lower is preferred. This is because if the height of the first partition is higher than the height of the second partition, it is difficult to seal the cell culture container with a lid. On the other hand, the first partition is configured such that the first storage portion formed by the inner bottom surface and the first partition has a height that can store a sufficient amount of cells and culture medium. The height of the first partition is, for example, 5 to 20 mm.

  The first partition is detachably attached to the inner bottom surface of the container. Although it does not restrict | limit especially as a detachable attachment method, The method of attaching through an adhesive agent is mentioned. As the adhesive, those having biocompatibility are preferable. Moreover, what can adhere | attach with the adhesive strength of the grade which can be attached or detached by applying external force is preferable. For example, fibrin adhesives, cyanoacrylate adhesives, gelatin adhesives obtained by crosslinking gelatin with formaldehyde or glutaraldehyde, polyurethane adhesives, adhesives containing biodegradable polymers (albumin, collagen, gelatin, etc.) Agents (see, for example, Japanese Patent No. 4844806) and adhesives described in Hayashi, Journal of the Japan Welding Society, Vol. 70, pages 19 to 23, 2001, and the like. Moreover, as a detachable attachment method, a method in which the first partition and the inner bottom surface of the container are fitted to each other may be used.

  The material constituting the first partition is not particularly limited, and may be the same material as the material constituting the inner bottom surface and the second partition, and in one embodiment, the material constituting the culture dish. In one embodiment, the first partition is preferably formed of an elastomer material. By using an elastomer material for the first partition wall, the first partition wall can be detachably attached to the inner bottom surface of the container simply by pressure bonding without using an adhesive. In that case, the material which comprises the inner bottom face used as the object which arrange | positions a 1st partition is made into glass or plastic, and the adhesiveness in the case of crimping | bonding a 1st partition and an inner bottom face can be increased.

  The elastomer material includes a rubber material, a thermosetting resin-based elastomer material, and a thermoplastic resin-based elastomer material, and a thermosetting resin-based elastomer material is preferable from the viewpoint of heat resistance. The rubber material includes natural rubber and synthetic rubber (polybutadiene rubber, nitrile rubber, chloroprene rubber). Specific examples of the elastomer material include urethane rubber, nitrile rubber, silicone rubber, silicone resin (for example, polydimethylsiloxane), fluorine rubber, acrylic rubber, isoprene rubber, ethylene propylene rubber, chlorosulfonated polyethylene rubber, epichlorohydrin rubber, chloroprene. Examples thereof include rubber, styrene / butadiene rubber, butadiene rubber, and polyisobutylene rubber. Silicone rubber and silicone resin are particularly preferable because they are excellent in heat resistance and stable as a solid. Silicone rubber and silicone resin are also suitable in that the elasticity can be adjusted by the amount of the curing agent.

  Even when an elastomer material is used as the first partition, the first partition and the inner bottom surface can be more stably fixed using an adhesive. In that case, it is preferable to use an adhesive made of the same or the same material as that constituting the first partition as the adhesive. For example, when the first partition made of silicone rubber or silicone resin is fixed, it is preferable to use a silicone rubber adhesive or a silicone resin adhesive. If it is the said combination, while being able to fix stably by the effect of an adhesive agent, attachment or detachment of a 1st partition can also be implemented easily.

  Although the inner bottom surface of the cell culture container of the present invention is non-cell-adhesive, the cell contact region on the other inner surface of the cell culture container is also non-cell-adhesive, from the viewpoint of ease of peeling of the cell laminate To preferred. For example, the surface of the first partition, the surface of the second partition, or the inner surface of the culture dish forming the inner bottom surface and the second partition are also preferably non-cell-adhesive. Cell adhesion and cell non-adhesion indicate a relative relationship between the degree of cell adhesion in one region and the other region. Cell adhesion means that cells are easily adhered. Cell adhesion is determined by whether or not cell adhesion or extension is likely to occur depending on the chemical or physical properties of the surface.

As an index for determining cell adhesion, the cell adhesion spreading rate when cells are actually cultured can be used. The cell adhesive surface is preferably a surface having a cell adhesion extension rate of 60% or more, and more preferably a surface having a cell adhesion extension rate of 80% or more. If the cell adhesion extension rate is high, cells can be cultured efficiently. The cell adhesion extension rate in the present invention is such that cells to be cultured within a seeding density of 4000 cells / cm ² or more and less than 30000 cells / cm ² are seeded on the surface to be measured, and in an incubator at 37 ° C. and a CO ₂ concentration of 5%. It is defined as the ratio of cells that have adhered and spread when cultured for 14.5 hours ({(number of cells adhered) / (number of cells seeded)} × 100 (%)).

  On the other hand, cell non-adhesiveness refers to the property that cells are difficult to adhere. Cell non-adhesiveness is determined by whether or not cell adhesion or extension is unlikely to occur due to the chemical or physical properties of the surface. The non-cell-adhesive surface is preferably a surface having a cell adhesion extension rate as defined above of less than 60%, more preferably less than 40%, and even more preferably 5% or less. The surface is preferably 2% or less, and most preferably.

  The cell culture container in which the inner surface including the inner bottom surface is non-cell-adhesive is, for example, the first partition wall, the second partition wall, and the surface of the material that personalizes the inner bottom surface, or in one embodiment, the inner bottom surface. And it can obtain by making the surface of the material which comprises the culture dish which comprises the 2nd partition into a hydrophilic surface by performing a hydrophilic treatment.

  The hydrophilization treatment can be performed by a method usually used in the art, and is not particularly limited. For example, plasma treatment, coating treatment, UV irradiation treatment, EB irradiation treatment, graft polymerization treatment of hydrophilic polymer on the surface, etc. are mentioned, but the shape of the treatment object has a three-dimensional fine and complicated structure. However, plasma treatment is preferable from the viewpoint of uniformly treating the whole.

Examples of the plasma treatment include plasma treatment using an inert gas. Examples of the inert gas include noble gases such as helium (He), neon (Ne), argon (Ar), and xenon (Xe), nitrogen (N ₂ ), oxygen (O ₂ ), and fluorocarbon gas. . Examples of the fluorocarbon include unsaturated fluorocarbons (fluorocarbons having an unsaturated bond) such as fluoroethylene (C ₂ F ₄ ) and hexafluoropropylene (CF ₃ CFCF ₂ ), carbon tetrafluoride ( Saturated fluorocarbons such as CF ₄ ), carbon hexafluoride (C ₂ F ₆ ), octafluorocyclobutane (C ₄ F ₈ ), trifluoromethane (CHF ₃ ), and octafluoropropane (C ₃ F ₈ ) Can be mentioned. As carbon fluoride, ethylene trichloride trichloride (C ₂ ClF ₃ ), carbon trichloride monofluoride (CCl ₃ F), carbon monobromide trifluoride (CBrF ₃ ), hexafluoroacetone ((CF ₃ ) ₂ CO), hexafluoroalcohol ((CF ₃ ) ₂ CHOH) and other halogen containing elements other than fluorine may be used. Plasma treatment using oxygen gas or argon gas is preferable from the viewpoint of maintaining hydrophilicity for a long period of time. Further, from the viewpoint of preventing surface degradation (decomposition and etching) and the like, low-temperature plasma treatment generated under atmospheric pressure is preferable.

  Examples of the coating treatment include a coating treatment with a hydrophilic polymer. The hydrophilic polymer refers to a polymer containing a carbon component and having a hydrophilic functional group in the main chain or side chain of the polymer. The hydrophilic polymer is preferably a water-soluble polymer containing a carbon-oxygen bond and having water solubility and water swellability. The hydrophilic polymer may be constantly water-soluble or water-swellable, or may be water-soluble or water-swellable by a predetermined stimulus such as light, temperature, and pH.

  Specific examples of the hydrophilic polymer include polyalkylene glycol, polyvinyl pyrrolidone, polypropylene glycol, polyvinyl alcohol, polyethyleneimine, polyallylamine, polyvinylamine, polyvinyl acetate, polyacrylic acid, poly (meth) acrylamide, poly-2-hydroxy Examples thereof include methacrylate (poly-HEMA), copolymers of these and other monomers, and graft polymers such as 2-methacryloyloxyethyl phosphorylcholine polymer (MPC polymer). Among them, polyalkylene glycols having various molecular weights are commercially available and are excellent in biocompatibility, so that they can be suitably used. Specific examples of the polyalkylene glycol include polyethylene glycol (PEG), polypropylene glycol, and a copolymer of ethylene glycol and propylene glycol. In the present invention, polyethylene glycol is particularly preferably used as the hydrophilic polymer.

  By forming a cell culture vessel including the first partition wall, the second partition wall, etc. with a hydrophilic material such as the hydrophilic polymer as described above, the surface can be made hydrophilic even without a hydrophilic treatment. it can. Examples of other hydrophilic materials include polyacrylonitrile, nylon 6, polyethylene terephthalate, polymethyl acrylate, polymethyl methacrylate, and phenol resin.

  The hydrophilic surface means a surface having a water contact angle of 70 ° or less. The water contact angle of the hydrophilic surface is preferably 60 ° or less, more preferably 50 ° or less, still more preferably 45 ° or less, and preferably 10 ° or more. If a water contact angle is the said range, it can be said that it has sufficient hydrophilicity. The water contact angle refers to a water contact angle measured at 23 ° C.

  In the present invention, the non-cell-adhesive surface includes a surface capable of changing from cell-adhesive to non-cell-adhesive. Such a surface may be formed by immobilizing at least one stimulus-responsive material selected from the group consisting of a temperature-responsive material, a pH-responsive material, and an ion-responsive material on the surface of the substrate. it can. The stimulus-responsive material is particularly preferably a temperature-responsive polymer, but is not limited thereto.

  The temperature-responsive polymer that can be suitably used in the present invention is hydrophobic at the cell culture temperature (usually about 37 ° C.) and hydrophilic at the temperature at the time of recovery of the cultured cell laminate. . Specifically, the temperature-responsive polymer that can be suitably used in the present invention is preferably a polymer having a lower critical solution temperature T of 0 to 80 ° C, preferably 0 to 50 ° C. Such suitable polymers include acrylic or methacrylic polymers. Specifically, poly-N-isopropylacrylamide (T = 32 ° C.), poly-Nn-propyl acrylamide (T = 21 ° C.), poly-Nn-propyl methacrylamide (T = 32 ° C.), poly N-ethoxyethyl acrylamide (T = about 35 ° C.), poly-N-tetrahydrofurfuryl acrylamide (T = about 28 ° C.), poly-N-tetrahydrofurfuryl methacrylamide (T = about 35 ° C.), and poly- N, N-diethylacrylamide (T = 32 ° C.) and the like can be mentioned.

  In the cell culture container of the present invention, the first partition wall is not disposed on the inner bottom surface, that is, the container in which the second partition wall is disposed on the inner bottom surface (in one embodiment, the inner bottom surface and the second partition wall). A combination form of a culture dish constituting the partition wall and the first partition wall is also included (for example, a combination form shown by B1 and B2 in FIG. 1).

The present invention also relates to a method for producing a cell laminate using the above-described cell culture container. The method for producing a cell laminate of the present invention includes, for example, as shown in FIG.
a) preparing the cell culture container 1 described above,
b) a step of adding the cells 7 and the culture medium 8 to the first container 5;
c) performing cell culture while applying centrifugal force 9 toward the inner bottom surface to form the cell stack 10;
d) a step of adding the culture medium 8 to the second container 6;
e) removing the first partition; and f) culturing the cell stack 10.

  The cells used in the present invention are not particularly limited as long as they are adherent cells. Examples of such cells include liver cells that are liver parenchymal cells, Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, Epidermal cells such as epidermal keratinocytes, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells such as corneal epithelial cells, mammary cells, pericytes, muscle cells such as smooth muscle cells and cardiomyocytes, kidneys Examples include cells, pancreatic islets of Langerhans, nerve cells such as peripheral nerve cells and optic nerve cells, and bone cells such as chondrocytes.

  These cells may be primary cells collected directly from tissues or organs, or may be passaged from one generation to another. Furthermore, these cells are undifferentiated embryonic stem cells, pluripotent stem cells such as pluripotent mesenchymal stem cells, unipotent stem cells such as vascular endothelial progenitor cells that have unipotency, and differentiation has ended. Any of cells may be sufficient. In addition, the cells may be cultivated as a single species, or two or more types of cells may be co-cultured.

  These cells are cultured in advance by a conventional method, proliferated, and detached by enzyme treatment. As the medium, any cell culture medium that is usually used in the art can be used without particular limitation. For example, depending on the type of cells used, MEM medium, BME medium, DME medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI1640 medium, etc. A basal medium as described in “Tissue culture technology third edition”, page 581, edited by the Japanese Society for Tissue Culture can be used. Furthermore, serum (such as fetal bovine serum), various growth factors, antibiotics, amino acids and the like may be added to the basal medium. A commercially available serum-free medium such as Gibco serum-free medium (Invitrogen) can also be used. Since the adherent cells grow by adhering to the surface of the culture container during culture, they can be recovered by detaching them from the culture container by enzyme treatment and suspending them in the medium.

  Step b) is a step of adding the cells thus collected together with the culture medium to the first accommodating portion of the cell culture container. Here, when the cells peeled and collected by the enzyme treatment are added to the first container of the cell culture container, it is preferable to resuspend the cells in the medium and add the cells. The medium in which the cells are suspended is not particularly limited, and can be selected according to the type of cells such as the above-mentioned various media and buffers. For example, phosphate buffer buffer (PBS), Hanks buffer solution (HBSS), etc. are mentioned. A medium that is optimal for cell survival is preferred. However, from the viewpoint of cell survival, the pH is preferably in the range of 7.0 to 7.6, more preferably 7.2 to 7.4. Moreover, you may improve the intensity | strength of a cell laminated body by further adding cell adhesion protein to a cell suspension. From the viewpoint of improving cell viability, animal-derived serum may be added to the cell suspension.

  Step c) is a step of forming a cell stack by performing cell culture while applying a centrifugal force toward the inner bottom surface of the cell culture container. In this step, cells adhere to each other in a state where the cells are in close contact with the inner bottom surface according to the shape of the inner bottom surface by centrifugal force, and a tissue having a desired shape is formed.

  The inner bottom surface direction is preferably a direction perpendicular to the inner bottom surface. The direction perpendicular to the inner bottom surface is not intended to be strictly perpendicular to the inner bottom surface. For example, there may be a deviation within 30 ° with respect to the strictly perpendicular direction.

  The magnitude of the centrifugal force can be appropriately selected as long as the tissue can be formed without adversely affecting the function of the cells. The centrifugal force is preferably applied at 2 to 2000 G, more preferably 10 to 1000 G, and more preferably 50 to 800 G. In centrifugation, a culture vessel containing a cell suspension is placed in a centrifuge and centrifugal force can be applied by performing centrifugation.

  The centrifugation time can also be appropriately selected as long as the tissue can be formed without adversely affecting cell functions. Preferably it is 5 minutes or more, More preferably, it is 60 minutes or more, More preferably, it is 90 minutes or more, Preferably it is within 840 minutes, More preferably, it is within 720 minutes, More preferably, it is within 360 minutes.

  The cell culture is preferably performed under conditions where cell-cell adhesion is formed. “Conditions where intercellular adhesion is formed” refers to conditions under which cells can act and adhere to each other. Although it varies depending on the type of cells to be cultured, for example, the temperature condition is preferably 20 to 40 ° C., and the atmospheric gas condition is preferably a carbon dioxide concentration of 3 to 5%.

  It is sufficient that the adhesion between cells is formed in the centrifugal treatment, and it is not essential to increase the number of cells by proliferation. By appropriately adjusting the number of cells in the medium, it is possible to control the thickness of the produced cell stack (that is, the number of cell stacks in the thickness direction). Since it is not necessary to proliferate cells in the centrifugation step, a cell laminate can be obtained in a relatively short time. In addition, the thickness and shape of the cell stack can be freely adjusted.

  After completion of the centrifugation, the obtained cell stack can be peeled off from the inner bottom surface of the cell culture container by a simple physical operation such as a pipetting operation. Since the inner bottom surface of the cell culture container used for the centrifugation treatment is non-adhesive, this operation is easy, and the cell stack can be produced and detached in a short time of culture. When the inner bottom surface is a surface that changes to cell non-adhesive properties such as a stimulus-responsive material, the peeling operation is performed in an environment in which the cells become non-adhesive (for example, a temperature below the lower critical temperature). The cell laminate obtained is preferably in the form of a cell sheet having a layer structure, more preferably a layer structure of three or more layers.

  Step d) is a step of adding a culture medium to the second storage unit. The medium to be added here may be the same as described above, but it contains nutrients necessary for cell survival because it aims to supplement nutrients for further culture of the cell stack. It is preferable that the medium is optimal for cell survival. Examples of nutrients necessary for cell survival include carbon sources such as glucose, nitrogen sources and inorganic ions, various amino acids and proteins.

  When adding a culture medium to a 2nd accommodating part, it is preferable to add in the quantity which becomes the height equivalent to the height of the liquid of the mixture of the cell accommodated in the 1st accommodating part and a culture medium. By doing so, when removing the first partition wall in step e), it is possible to suppress the shaking and vibration of the liquid and to prevent the cell stack from being damaged. In the cell laminate formed by the centrifugation in step c), cell-cell adhesion is formed, but it is not strong, so it is desirable to suppress damage as much as possible to avoid damage.

  Step e) is a step of removing the first partition. The removal of the first partition can be performed with tweezers or the like, but as described above, it is performed so as not to cause liquid shaking and vibration as much as possible so as not to damage the cell stack formed by centrifugation. It is preferable. By removing the first partition, the contents of the first storage part and the contents of the second storage part are mixed.

  Step f) is a step of further culturing the cell stack. Since the 1st partition is removed, a cell laminated body will be cultured in presence of the additional culture medium mixed from the 2nd accommodating part in a wider area | region. Therefore, even if nutrients and the like in the medium are consumed and insufficient in the formation of the cell laminate under the centrifugal treatment, the nutrients and the like contained in the medium added to the second storage unit are added to the cells. The survival of the cells contained in the laminate can be maintained. Furthermore, cell culture can be promoted by the added nutrient, and the formation of cell-cell adhesion can also be promoted.

  The culture condition of the cell laminate in step f) is not particularly limited, but it is preferably performed under the condition that cell-cell adhesion is formed as described above. For example, the temperature condition is preferably 20 to 40 ° C., and the atmospheric gas condition is preferably a carbon dioxide concentration of 4 to 6%. The culture time is not particularly limited, but is preferably 12 to 48 hours, for example. Adequate cell-cell adhesion can be formed by setting it to 12 hours or longer, and cell necrosis due to consumption of medium components can be prevented by setting it to 48 hours or shorter.

  In the method of the present invention, the centrifuge treatment and the culture may be repeated by repeating steps c) to f) using a cell culture vessel having a further partition and a further accommodating portion. For example, a cell culture vessel having a third partition wall outside the second partition wall and having a third housing portion formed by the inner bottom surface, the second partition wall, and the third partition wall can be used. For example, after culturing the cell laminate in step d) to enhance cell-cell adhesion, preferably more cells are added, and c) is repeated by repeating step c) to obtain a larger cell laminate. Can be formed. Then, the step d) is repeated to add the medium to the third accommodating part, the step e) is repeated to remove the third partition wall, and the step f) is repeated to culture the cell stack, thereby intercellular adhesion. Can be strengthened. The same applies to the case of using a cell culture container having a fourth partition wall outside the third partition wall.

  In the method of the present invention, there is no need to transfer the cell stack to another cell culture vessel for culturing the cell stack after centrifugation, and the cell stack is broken during the transfer. Can be avoided. If the culture is continued as it is without transferring the cell stack to another cell culture vessel, the problem that the amount of the medium is insufficient relative to the number of cells and the cells die can be avoided. If the medium is simply added with a pipette or the like to make up for the lack of medium, the cell stack may be damaged by vibration or liquid shaking. However, according to the present invention, the medium is added without damaging the cell stack. be able to.

  According to the present invention, it is possible to obtain a cell laminate in which cells are densified. Furthermore, the cell laminate obtained by the present invention has improved cell-cell adhesion and has a strength that can be easily recovered. Therefore, it is difficult to break during handling and is preferable for transplantation.

  Moreover, the cell laminated body of desired size can be manufactured by changing the size of a 1st partition suitably.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the scope of the present invention is not limited to the range of an Example. In Examples,% means mass% unless otherwise specified.

<Example 1>
Polydimethylsiloxane KE-1830 and catalyst CAT-1300L-3 (both Shin-Etsu Chemical Co., Ltd.) were mixed at a weight ratio of 10: 1 and then stirred. Thereafter, the mixture was poured into a silicone mold and degassed, and then cured by heating in an oven at 75 ° C. for 90 minutes.

  After curing, the first partition peeled from the mold was adhered to the bottom of a polystyrene 3.5 cm Petri dish (Becton Dickinson) using the same polydimethylsiloxane as the adhesive, and the oven was placed under the same conditions as above. It was cured by heating.

  A photograph of the prepared culture vessel is shown in FIG. The diameter of the inner periphery of the first partition wall was 18 mm, and the diameter of the inner periphery of the second partition wall (petri dish side wall) was 28 mm. The first partition wall was removable by applying force with tweezers or the like.

<Test Example 1>
A cell stack was prepared using the culture vessel prepared in Example 1. The culture container prepared in Example 1 was used after being sterilized with 70% ethanol and washed with a phosphate buffer buffer (PBS).

CCL163 cells (mouse fibroblasts) grown using DMEM medium (Sigma) containing 10% fetal bovine serum were collected by trypsin treatment. 1 × 10 ⁷ cells were seeded inside the first partition of the culture vessel prepared in Example 1, that is, in the first container, and the conditions were 37 ° C. and 5% CO ₂ concentration in the same medium as above. Then, the cells were layered by centrifugation at 1000 rpm for 3 hours. Cell stacking could be carried out without leakage of the medium.

  In Example 2, the cell stack was peeled off by pipetting the medium after completion of the centrifugation. Subsequently, the medium is filled between the first partition wall and the dish side wall, that is, the second container, and then the first partition wall is removed using sterilized tweezers and cultured at 37 ° C. for 15 hours. It was. A photograph of the resulting cell stack is shown in FIG.

  In Comparative Example 1, after completion of the centrifugation, the culture was continued without removing the first partition (culture at 37 ° C. for 15 hours).

  In Comparative Example 2, using a 3.5 cm Petri dish without the first partition, a cell stack was prepared by the same centrifugation as described above, and after completion of the centrifugation, 2 ml of the medium was added, and the culture was continued at 37 ° C. for 15 hours. Was cultured.

  In Comparative Example 3, a cell stack was prepared by the same centrifugal treatment as described above using a 3.5 cm Petri dish having no first partition, and the obtained cell stack was 6 cm filled with about 9 ml of a medium. It moved to the petri dish and culture | cultivated for 15 hours at 37 degreeC.

In Comparative Examples 2 and 3, 3 × 10 ⁷ CCL163 cells were seeded so that the cell density was almost the same as in the Examples, and then centrifuged in the same manner to stack the cells.

  A summary of the operations of Example 2 and Comparative Examples 1 to 3 is shown in the table of FIG.

  In Comparative Examples 1 and 2, although the cell stack was recovered by pipetting the medium, the medium turned yellow in the culture after centrifugation, and holes were formed in the cell stack sheets. This is probably because the amount of the medium was small relative to the number of cells, so that nutrients were insufficient and the cells were killed.

  In Comparative Example 3, the cell laminate was recovered by pipetting the medium, but tearing was observed in the cell laminate sheet when transferred to a 6 cm Petri dish.

  A summary of the results is shown in the table of FIG. In the table, ○ indicates that the cell stack was recovered without defect, △ indicates that the cell stack was recovered, but there was a defect such as tearing, and × indicates that the cell stack was separated. Indicates that recovery was not possible.

<Test Example 2>
In order to observe the state of cell-cell adhesion of the cell laminate produced in Example 2 of Test Example 1, the following analysis was performed.

  The cells were fixed by immersing the cell laminate in a 4% paraformaldehyde solution at room temperature for 20 minutes. Thereafter, the sample was placed on a filter paper, embedded with paraffin substitution, a 5 μm-thick section was prepared with a microtome, and stained with HE (hematoxylin and eosin). The specific protocol was performed according to the method described in the data attached to each staining reagent (Wako Pure Chemical Industries, Ltd.).

  As Comparative Example 4, the cell laminate immediately after centrifugation was collected and similarly subjected to HE staining.

  The results are shown in FIG. From this, when the cells were stacked as in Comparative Example 4 and additional culture was not performed, there were many voids inside, whereas additional culture was performed after centrifugation as in Example 2. In the case, it was found that cell-cell adhesion is dense. Cell survival was also confirmed.

<Test Example 3>
The 1st partition produced similarly to Example 1 was fixed by crimping | bonding to the bottom face of a 3.5cm Petri dish made from polystyrene (Becton Dickinson), without using polydimethylsiloxane.

  Subsequently, 1 ml of DMEM medium was put into the first partition, and after confirming that there was no leakage, centrifugation was performed under the conditions described in Test Example 1 to examine whether the medium leaked or the first partition moved. .

  As a result, no leakage of the medium or displacement of the first partition was observed. From this, it was found that fixing with an adhesive is not essential when the first partition made of an elastomer material is used.

1: Cell culture vessel 2: Inner bottom surface 3: Second partition wall (side wall of culture dish)
4: 1st partition 5: 1st accommodating part 6: 2nd accommodating part 7: Cell 8: Medium 9: Centrifugal force 10: Cell laminated body

Claims (5)

A cell culture container for producing a cell laminate,
A cell non-adherent inner bottom surface, and a first partition wall and a second partition wall disposed on the inner bottom surface,
The inner bottom surface and the first partition form a first accommodating part capable of accommodating cells and a medium,
The inner bottom surface, the first partition wall, and the second partition wall form a second storage portion that can store cells and a culture medium,
The first partition has a cylindrical shape penetrating from one end to the other end,
The first accommodating portion is closed by the inner bottom surface on one end side of the first partition, and is opened on the other end side of the first partition,
The entire inner bottom surface of the first housing portion that closes one end of the first partition wall is exposed through the opening on the other end side of the first partition wall,
The second partition wall surrounds the entire outer periphery of the second housing portion, and the first partition wall is disposed inside the second partition wall.
The cell culture container, wherein the first partition wall is detachably attached to the inner bottom surface. The second partition is cylindrical, the cell culture vessel according to claim 1.   The cell culture container according to claim 1 or 2, wherein the first partition is formed of an elastomer material. A method for producing a cell laminate,
a) preparing a cell culture container,
The cell culture container includes a cell non-adhesive inner bottom surface, and a first partition wall and a second partition wall disposed on the inner bottom surface,
The inner bottom surface and the first partition form a first accommodating part capable of accommodating cells and a medium,
The inner bottom surface, the first partition wall, and the second partition wall form a second storage portion that can store cells and a culture medium,
The second partition wall surrounds the entire outer periphery of the second housing portion, and the first partition wall is disposed inside the second partition wall.
The first partition is detachably attached to the inner bottom surface, and b) the step of adding cells and culture medium to the first accommodating portion;
c) performing cell culture while applying centrifugal force toward the inner bottom surface to form a cell stack,
d) adding a culture medium to the second container;
e) The method comprising the steps of removing the first partition, and f) culturing the cell stack. First partition and the second partition wall is tubular, the method of claim 4.

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