JP2016111975A - Cell culture vessel and a method for producing cell laminate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a cell laminate in which a cell laminate produced by using centrifugal force can be easily recovered and transferred without breaking a shape of the laminate.SOLUTION: A method for producing a cell laminate has: a cell addition process of inputting culture solution containing a cell into a cell culture vessel 1 that has a bottom part 10 having an inner surface 101 which is cell non-adhesive or capable of changing to cell non-adhesive, and an upper part 20 opposing to the bottom 10, and in which the upper part 20 has 200 cc/mday atm or less of oxygen permeability and is separably constituted; a cell culture process of performing cell culture under the condition that intercellular adhesion is formed while centrifugal force in a direction of the inner surface 101 of the bottom part 10 is allowed to work to the cell, and forming a cell laminate by adhering cells; a mounting process of inverting positions of the bottom part 10 and the upper part 20 to peel an obtained cell laminate from the inner surface 101 and mounting the laminate onto the upper part 20; and a separation process of separating the upper part 20 together with the mounted cell laminate.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a cell culture container and a method for producing a cell laminate using the same.

  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.

  As a method for easily producing a multi-layered cell sheet (cell laminate), (Patent Document 1) includes a cell non-adhesive property or a cell non-adhesive property. A cell addition step in which a culture solution containing cells is added to a culture vessel having a bottom surface, and a condition in which cell-cell adhesion is formed while a centrifugal force is applied to the cells added to the culture vessel toward the inner bottom surface. Disclosed is a method for producing a tissue, which includes a cell culturing step of forming a tissue by performing cell culture and adhering cells, and a detaching step of detaching and collecting the tissue obtained in the cell culturing step from the inner bottom surface. ing.

  In this method, it is possible to produce a living tissue in a shorter time while easily controlling the number of cell layers of the tissue, but because an enzyme is used to peel off the cells grown in the culture solution. In addition, there was room for improvement in handling properties because the cell-cell bond in the prepared tissue was weak and the strength of the tissue was weak. Accordingly, it has been desired to develop a container that can be easily transferred without destroying the tissue of the cell laminate produced using centrifugal force, and a method for producing the cell laminate using the container.

Japanese Patent No. 5407343

  In view of the above-described conventional situation, the present invention provides a cell culture container capable of easily transferring a cell laminate produced using centrifugal force without losing its shape, and a method for producing a cell laminate using the same. For the purpose.

  In order to solve the above-mentioned problems, the present inventors have conducted intensive research. As a result, the upper part of the cell culture vessel has a predetermined oxygen permeability and is separable, and a cell stack is placed on the upper part. Thus, it was found that the handling property of the cell laminate was improved by the transfer, and the invention was completed. That is, the gist of the present invention is as follows.

(1) It has a bottom portion having an inner surface that is non-cell-adhesive or can be changed to cell-non-adhesion, and an upper portion facing the bottom portion, and the upper portion is 200 cc / m ² · day. A cell culture container having an oxygen permeability of atm or less and configured to be separable.
(2) The cell culture container according to (1), wherein the oxygen permeability is 15 cc / m ² · day · atm or less.
(3) The cell culture container according to (2), wherein the upper part is a polyethylene terephthalate film or a laminated film containing the polyethylene terephthalate film.
(4) It has a bottom portion having an inner surface that is non-cell-adhesive or can be changed to cell-non-adhesion, and an upper portion facing the bottom portion, and the upper portion is 200 cc / m ² · day. A cell addition step of placing a culture solution containing cells in a cell culture vessel having an oxygen permeability of atm or less and configured to be separable;
A cell culturing step of performing cell culture under a condition in which cell-cell adhesion is formed while acting centrifugal force toward the inner surface of the bottom portion on the cells, and bonding the cells to form a cell stack,
Reversing the positions of the bottom and the top, and separating the resulting cell stack from the inner surface and placing it on the top;
A separation step of separating the upper part together with the placed cell stack;
A method for producing a cell laminate comprising:

  According to the present invention, a cell stack produced using centrifugal force can be easily recovered and transferred without causing destruction, deformation, or the like.

It is the side sectional view (a) and top view (b) which show one embodiment of the cell culture container concerning the present invention. It is a figure for demonstrating the cell culture process in the preparation methods of the cell laminated body which concerns on this invention. It is a figure for demonstrating the mounting process in the preparation methods of the cell laminated body which concerns on this invention. It is a figure for demonstrating the isolation | separation process in the preparation methods of the cell laminated body which concerns on this invention.

  Hereinafter, the present invention will be described in detail based on embodiments.

  The method for producing a cell laminate of the present invention has a bottom portion having an inner surface that is non-cell-adhesive or capable of changing to cell-non-adhesion, and an upper portion that faces the bottom, and the upper portion is A cell addition step of putting a culture solution containing cells into a cell culture vessel that has a predetermined oxygen permeability and is separable, and a centrifugal force acting on the cells toward the inner surface of the bottom. A cell culture step in which cell-cell adhesion is formed under the condition that cell-cell adhesion is formed, and cell stacks are formed by adhering cells, and the positions of the bottom and top are reversed, and the resulting cell stack is And a separation step of separating the upper portion together with the placed cell stack, and a separation step of separating the upper portion together with the placed cell stack.

1. Cell Addition Step First, the configuration of a cell culture container for containing a culture solution containing cells in the cell addition step will be described. FIG. 1 shows an embodiment of a cell culture container. The cell culture container 1 in FIG. 1 has a bottom portion 10 having an inner surface 101 and an upper portion 20 facing the bottom portion 10. In this embodiment, the bottom 10 is configured as a part of a petri dish-like container member 11 having a side wall. The upper portion 20 is laminated on the upper surface of the lid member 21 combined with the container member 11. The lid member 21 has a through hole 210 formed at the center thereof, the through hole 210 is covered with the upper part 20, and the upper part 20 is exposed to the internal space of the cell culture container. In addition, it is not limited to embodiment of FIG. 1, For example, the upper part 20 serves as the cover member 21, and the film-form or plate-shaped upper part 20 is directly laminated | stacked on the upper surface of the petri dish-shaped container member 11, and the container member 11 of FIG. It is good also as a structure which covers an opening part.

  Although there is no restriction | limiting in particular regarding the planar view area of the container member 11, the cover member 21, and the upper part 20, It is preferable that it is larger than the cell laminated body to produce. Further, the planar shape of the container member 11, the lid member 21 and the upper portion 20 is not particularly limited, and may be a polygonal shape instead of a circular shape. However, it is circular from the viewpoint of ease of manufacture and ease of handling. Preferably there is. Furthermore, the container member 11, the lid member 21 and the upper part 20 constituting the cell culture container 1 are preferably subjected to sterilization in advance in order to handle cells. There is no particular limitation on the sterilization method, and various methods such as ethanol sterilization, UV irradiation, EB irradiation, ethylene oxide gas sterilization, and γ-ray irradiation can be used.

  The upper part 20 can be in the form of a film or a plate, but in consideration of the handling property when the cell laminate produced thereon is transferred and used for transplantation work or the like, flexibility can be obtained. It is preferable that the resin film is transparent, and it is preferable to be transparent in order to visually recognize the cells. The thickness of the upper portion 20 is not particularly limited as long as it has a predetermined oxygen permeability. However, if it is too thin, handling properties when transporting together with the cell stack decreases, and centrifugal force However, if it is too thick, it takes time to separate from the lid member 21, so that it is appropriately set in consideration of these balances. Specifically, when the upper portion 20 is formed of a resin film, it varies depending on the type of resin, but is preferably in the range of 30 μm to 100 μm, particularly preferably 40 μm to 80 μm.

The upper portion 20 needs to have an oxygen permeability of 200 cc / m ² · day · atm or less. It is preferably 15 cc / m ² · day · atm or less. If oxygen permeability is high, the oxygen concentration in the culture medium is higher than that in the case of using a normal culture dish, and this may cause cytotoxicity (Forsyth et al., Aging Cell). , Vol. 2, 235-243, 2003). In particular, when a cell laminate is formed using centrifugal force, maintaining the pH of the culture solution by controlling the CO ₂ concentration is important for improving the cell viability. If the oxygen permeability of the upper part 20 is high, there is no problem if the centrifuge is constantly supplied with CO _2, but if this is not the case, the CO ₂ concentration of the culture solution becomes almost the same as the outside air, Cell death may occur due to a decrease in CO ₂ concentration and a shift of the culture medium to alkaline. Therefore, by controlling the oxygen permeability of the upper part 20 to 200 cc / m ² · day · atm or less, it is possible to maintain the gas concentration in the cell culture vessel during centrifugation, and a special type that supplies CO ₂ from the outside. Even without using the centrifuge, it is possible to produce a good cell laminate without reducing the cell viability.

  The material of the upper part 20 is not particularly limited as long as it satisfies the above oxygen permeability condition. When a resin film is used as the upper portion 20, as a specific example, a film of polyethylene terephthalate (PET), ethylene / vinyl alcohol copolymer resin (EVOH), polyvinylidene chloride, nylon, cellophane, or the like is preferably used. Polypropylene (PP) and polystyrene (PS) films are also applicable because they have a relatively high oxygen permeability but are within the above range.

Further, when a resin film is used as the upper portion 20, the resin film may be a single layer, or a laminated film obtained by laminating a plurality of resin films may be used. Polyethylene (PE) film, when used in a single layer, may have an oxygen permeability exceeding 200 cc / m ² · day · atm, but it can be used as a laminated film to increase barrier properties by increasing the thickness. Can be used as the upper part 20. As the upper portion 20, a polyethylene terephthalate film or a laminated film including a polyethylene terephthalate film is particularly preferably used.

  There is no restriction | limiting in particular about the adhesion method of the upper part 20 and the cover member 21, Various methods, such as the adhesion method using a solvent, the adhesion method by an adhesive agent, and a heat seal, can be employ | adopted suitably. In addition, when using an adhesive agent, it is desirable that the adhesive agent is comprised from the component which has biocompatibility. Further, the adhesive force is not particularly limited as long as the upper portion 20 can be separated after the cell laminate is formed. Specifically, it is desirable to be within a range of 3N to 6N / 15 mm in accordance with JIS Z0238.

  The cell culture container 1 used in the present invention may be configured so that at least the inner surface 101 of the bottom 10 is non-cell-adhesive or can be changed to cell-non-adhesive, Of the inner surface of the container member 11 that contains the culture medium containing cells, all of the surfaces that come into contact with the cells (for example, the inner bottom surface and the inner surface of the container member 11) are non-cell-adherent or non-cell-adherent. It is preferable to be able to change to sex.

  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, kidney cells And pancreatic islets of Langerhans, nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, bone cells and the like. These cells may be primary cells collected directly from tissues or organs, or may be obtained by substituting them for several generations. 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 is completed. Any of the cells may be used. The cells may be cultivated as a single species, or two or more cells may be co-cultured.

  These cells are cultured in advance by a conventional method, the culture is treated with trypsin treatment or the like, and is stored in the cell culture vessel 1 in a state of being suspended in the culture solution. Any culture medium can be used without particular limitation as long as it is a cell culture medium usually used in the art. 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 (fetal bovine serum etc.), various growth factors, antibiotics, amino acids, etc. may be added to the basal medium. A commercially available serum-free medium such as Gibco serum-free medium (Invitrogen) can also be used. Considering the clinical application of the finally obtained cell laminate, it is preferable to use a medium that does not contain animal-derived components.

In the present invention, “cell non-adhesive” means non-adhesive to cells to be cultured, and cell non-adhesive means a 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. Specifically, the cell adhesion spreading rate when actually culturing cells is used as an index, and a cell adhesion spreading rate of less than 60% is defined as cell non-adhesive. The cell adhesion extension rate is preferably less than 40%, more preferably 5% or less, and most preferably 2% or less. Here, the cell adhesion extension rate is determined by seeding cells to be cultured in a seeding density within a range of 4000 cells / cm ² or more and less than 30000 cells / cm ^{2 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 (%)). Therefore, depending on the cell type, a dish for suspension culture such as a petri dish can be used as it is. Alternatively, a surface treatment that becomes non-cell-adhesive can be applied as appropriate. In the case of cells with strong adhesive force (for example, canine kidney tubular epithelial cells (MDCK)), if the Petri dish is used as it is, the cell stack may adhere to the inner surface of the bottom, making recovery difficult. It is preferable to apply a surface treatment to form a non-cell-adhesive surface.

  Examples of the cell non-adhesive surface include a hydrophilic surface, specifically, a surface having a static water contact angle at 20 ° C. of 45 ° or less. However, a surface whose cell adhesion is enhanced by hydrophilic treatment such as plasma treatment or corona treatment is not preferable. The surface properties can be examined by measuring the surface O component by means such as elemental analysis and estimating the amount of hydroxyl groups on the surface. A non-cell-adhesive hydrophilic surface can be obtained by forming a film of an organic compound having a carbon-oxygen bond on the inner surface 101 of the bottom 10. Or you may comprise the bottom part 10 itself with the material which has hydrophilic property.

  The carbon-oxygen bond means a bond formed between carbon and oxygen, and is not limited to a single bond but may be a double bond. Examples of the carbon oxygen bond include a C—O bond, a C (═O) —O bond, and a C═O bond.

  Examples of main raw materials for the hydrophilic film include hydrophilic organic compounds such as water-soluble polymers, water-soluble oligomers, water-soluble organic compounds, surfactants, and amphiphilic substances. These are physically or chemically cross-linked with each other and physically or chemically bonded to the bottom 10 as a base material to form a hydrophilic film.

  Specific water-soluble polymer materials include polyalkylene glycol and derivatives thereof, polyacrylic acid and derivatives thereof, polymethacrylic acid and derivatives thereof, polyacrylamide and derivatives thereof, polyvinyl alcohol and derivatives thereof, and zwitterionic polymers. And polysaccharides. Examples of the molecular shape include a straight chain, a branched one, and a dendrimer. More specifically, polyethylene glycol, a copolymer of polyethylene glycol and polypropylene glycol, poly (N-isopropylacrylamide), poly (N-vinyl-2-pyrrolidone), poly (2-hydroxyethyl methacrylate), poly (methacryloyl) (Oxyethylphosphorylcholine), a copolymer of methacryloyloxyethylphosphorylcholine and an acrylic monomer, dextran, heparin, and the like, but are not limited thereto.

  Specific water-soluble oligomer materials and water-soluble organic compounds include alkylene glycol oligomers and derivatives thereof, acrylic acid oligomers and derivatives thereof, methacrylic acid oligomers and derivatives thereof, acrylamide oligomers and derivatives thereof, and saponified vinyl acetate oligomers and the like Derivatives, oligomers consisting of zwitterionic monomers and their derivatives, acrylic acid and its derivatives, methacrylic acid and its derivatives, acrylamide and its derivatives, zwitterionic compounds, water-soluble silane coupling agents, water-soluble thiol compounds, etc. Can do. More specifically, ethylene glycol oligomer, (N-isopropylacrylamide) oligomer, methacryloyloxyethylphosphorylcholine oligomer, low molecular weight dextran, low molecular weight heparin, oligoethylene glycol thiol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene Examples include, but are not limited to, glycol, 2- [methoxy (polyethyleneoxy) -propyl] trimethoxysilane, triethylene glycol-terminated-thiol, and the like.

  The average thickness of the hydrophilic film is preferably from 0.8 nm to 500 μm, more preferably from 0.8 nm to 100 μm, further preferably from 1 nm to 10 μm, and most preferably from 1.5 nm to 1 μm. An average thickness of 0.8 nm or more is preferable because it is not easily affected by the inner surface 101 of the bottom 10. If the average thickness is 500 μm or less, coating is relatively easy.

  As a method for forming a hydrophilic film on the inner surface 101 of the bottom portion 10, a method of directly adsorbing the main raw material (hydrophilic organic compound or the like) of the hydrophilic film on the inner surface 101, or a coating with a hydrophilic organic compound or the like directly on the inner surface 101. A method of coating a hydrophilic organic compound or the like on the inner surface 101, followed by a crosslinking treatment, a method of forming a hydrophilic film in a multi-stage manner in order to improve the adhesion to the inner surface 101, and improving the adhesion to the bottom 10 For this purpose, a method of forming a base layer on the inner surface 101 and then coating with a hydrophilic organic compound or the like, a method of forming a polymerization start point on the inner surface 101, and then polymerizing a hydrophilic polymer brush can be exemplified.

  Among the above film forming methods, particularly preferable methods include a method of forming a hydrophilic film in a multi-stage manner, and a base layer is formed on the inner surface 101 in order to improve adhesion to the bottom 10, and then hydrophilic organic The method of coating a compound etc. can be mentioned. This is because using these methods makes it easy to improve the adhesion of the hydrophilic organic compound or the like to the bottom 10. In this specification, the term “bonding layer” is used. The bonding layer means a layer existing between the outermost hydrophilic coating layer and the bottom portion 10 when a coating of a hydrophilic organic compound or the like is formed in a multistage manner, and a base layer is provided on the inner surface 101. When a hydrophilic film layer is formed on the underlayer, the underlayer is meant. The binding layer is preferably a layer containing a material having a binding portion (linker). Examples of combinations of the linker and the functional group at the end of the material to be bonded to the linker include epoxy group and hydroxyl group, phthalic anhydride and hydroxyl group, carboxyl group and N-hydroxysuccinimide, carboxyl group and carbodiimide, amino group and glutaraldehyde, etc. Is mentioned. In each combination, any of them may be a linker. In these methods, before coating with a hydrophilic material, a bonding layer is formed on the inner surface 101 with a material having a linker. The density of the material in the bonding layer is an important factor that defines the bonding force. The density can be easily evaluated using the contact angle of water on the surface of the bonding layer as an index. For example, taking a silane coupling agent having an epoxy group at the end (epoxy silane) as an example, the water contact angle of the inner surface 101 to which epoxy silane is added is typically 45 ° or more, preferably 47 ° or more. Then, a surface having sufficient cell non-adhesiveness can be obtained by adding an ethylene glycol-based material or the like in the presence of an acid catalyst.

  The inner surface 101 of the bottom 10 of the cell culture vessel 1 used in the present invention is preferably non-cell-adhesive from the viewpoint of ease of peeling of the cell laminate, but as another form, the cell-adhesive property during cell culture. However, it may be a surface capable of changing to cell non-adhesiveness upon peeling. Such a surface has at least one stimulus-responsive polymer selected from the group consisting of a temperature-responsive polymer, a pH-responsive polymer, and an ion-responsive polymer immobilized on the inner surface 101 through a covalent bond. Can be formed. The stimulus-responsive polymer 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 formed cell laminate. The temperature at which the temperature-responsive polymer changes from hydrophobic to hydrophilic (the critical dissolution temperature (T) in water) is not particularly limited, but from the viewpoint of easy recovery of the cell stack, the cell culture temperature It is preferable that the temperature is lower. By including such a temperature-responsive polymer component, since cell scaffolds (cell adhesion surfaces) are sufficiently secured during cell culture, cell culture can be performed efficiently. On the other hand, at the time of recovery of the cell laminate, the cell laminate can be more easily recovered by changing the hydrophobic portion to hydrophilicity and separating the cell laminate from the bottom 10. In particular, a temperature-responsive polymer that exhibits hydrophilicity at a temperature lower than a predetermined critical dissolution temperature and exhibits hydrophobicity at a temperature equal to or higher than the same temperature is preferable. The critical solution temperature in such a temperature-responsive polymer is particularly called the lower critical solution temperature.

  The temperature-responsive polymer that can be suitably used in the present invention is specifically preferably a polymer having a lower critical solution temperature T of 0 ° C. to 80 ° C., preferably 0 ° C. to 50 ° C. It is not preferable that T exceeds 80 ° C. because cells may die. Moreover, if T is lower than 0 ° C., the cell growth rate is generally extremely reduced or the cells are killed, which is not preferable. Such suitable polymers include acrylic polymers or methacrylic polymers. Specifically, for example, 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 Examples include poly-N, N-diethylacrylamide (T = 32 ° C.). Examples of other polymers include poly-N-ethylacrylamide, poly-N-isopropylmethacrylamide, poly-N-cyclopropylacrylamide, poly-N-cyclopropylmethacrylamide, poly-N-acryloylpyrrolidine, and poly-N. -Polyalkylene oxide block copolymers represented by alkyl-substituted cellulose derivatives such as acryloyl piperidine, polymethyl vinyl ether, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, block copolymers of polypolypropylene oxide and polyethylene oxide, An alkylene oxide block copolymer is mentioned.

  Examples of the monomer for forming these polymers include a monomer having a monomer homopolymer having T = 0 ° C. to 80 ° C. and capable of being polymerized by irradiation. Examples of the monomer include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, (meth) acrylamide derivatives having a cyclic group, and vinyl ether derivatives. More than seeds can be used. When one type of monomer is used alone, the polymer formed on the bottom 10 is a homopolymer, and when multiple types of monomers are used together, the polymer formed on the bottom 10 is a copolymer. Both forms are encompassed by the present invention. In addition, when it is necessary to adjust T depending on the type of proliferating cells, other monomers other than those described above may be further added for copolymerization. Further, a graft or block copolymer of the above polymer and another polymer, or a mixture of a temperature responsive polymer or the like and another polymer may be used. Moreover, it is also possible to crosslink within a range where the original properties of the polymer are not impaired.

  As the pH responsive polymer and the ion responsive polymer, those suitable for the cell laminate to be prepared can be appropriately selected.

2. Cell Culture Process In the cell culture process, cell culture is performed under the condition in which cell-cell adhesion is formed while acting a centrifugal force in the direction of the inner surface 101 of the bottom 10 on the cells in the culture solution accommodated in the cell culture container 1. To form a cell stack by adhering cells together. By this step, as shown in FIG. 2, the cell stack A is formed on the inner surface 101 of the bottom 10.

  The magnitude of the centrifugal force can be appropriately selected as long as the cell stack can be formed without adversely affecting the function of the cells. For example, the range of 2G-1440G is preferable, and the range of 2G-720G is more preferable. Centrifugal force can be applied by installing the cell culture container 1 containing the cell-containing culture solution in a centrifuge and performing a centrifugal operation.

  “Conditions under which cell-cell 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 ° C. to 40 ° C. The atmospheric gas condition is preferably a carbon dioxide concentration of 3% to 5%. 5 to 24 hours are preferred. By applying centrifugal force, the culture time can be shortened, and damage to cells can be reduced. The culture time can be shortened by separately preparing and adding an extracellular matrix, and the culture time can be 0.5 hours to 3 hours. When no extracellular matrix is added, the culture time can be 1 hour to 24 hours.

  It is sufficient that cell-cell adhesion is formed in the cell culture process, and it is not essential to increase the number of cells by proliferation. By appropriately adjusting the number of cells in the culture solution, it is possible to control the thickness of the produced cell stack (that is, the number of cell stacks in the thickness direction of the tissue). Since it is not necessary to proliferate cells in the cell culture process, a cell laminate can be obtained in a relatively short time. Moreover, the thickness and shape of the tissue of the cell stack can be freely adjusted.

  Moreover, it becomes possible to obtain the cell laminated body in which the cell was densified by utilizing a centrifugal force. A cell laminate in which cells are densified can be suitably used for transplantation.

3. Placement Step After the centrifugation operation, the placement step reverses the positions of the bottom 10 and the top 20 as shown in FIG. 3 and peels the obtained cell stack A from the inner surface 101 of the bottom 10 as well as the top 20. It is the process of mounting. If the inner surface 101 is a cell non-adhesive surface, the peeling operation is easy. If necessary, a tool such as tweezers is used as an auxiliary, or the cell stack A is moved to the upper 20 side by the action of gravity. You can make it. In addition, when the inner surface 101 is a surface that can be changed to non-cell-adhesive properties, such as a stimulus-responsive polymer, the environment (for example, a temperature that is lower than the lower critical temperature) becomes non-cell-adhesive. The peeling operation is performed in the state changed to).

4). Separation Step The separation step is a step of separating the upper portion 20 from the lid member 21 as shown in FIG. Prior to this, the culture solution remaining around the cell stack A placed on the upper portion 20 may be absorbed and removed by a pipette or the like as appropriate. The cell stack A is placed on the upper portion 20 by the separation step, and the cell stack A can be easily transferred to the site where, for example, cell transplantation is performed in this state. At that time, since the cell stack A is handled via the upper portion 20, it is possible to prevent the cell stack A from being destroyed or deformed.

  EXAMPLES Next, although an Example and a comparative example demonstrate this invention further in detail, the scope of the present invention is not limited to these Examples.

Example 1
Using a 3.5 cm sized Petri dish (manufactured by BD), on the lid of this Petri dish, an ultrasonic cutter was used to form a circular through-hole with an appropriate size (diameter: about 25 to 30 mm). The end was soaked with toluene so that the film could be adhered. Next, a biaxially stretched polystyrene film (OPS film; manufactured by Asahi Kasei Chemicals Co., Ltd.) was cut into a 35 mm size circle and adhered to the lid so as to cover the through hole, whereby the cell culture container of Example 1 was produced. This cell culture container was sterilized by performing UV irradiation in a clean bench.

Next, C2C12 cells of mouse myoblasts were grown in DMEM medium containing 10% fetal bovine serum (FBS), reached confluence, and then reacted with 0.01% trypsin for 10 minutes at 37 ° C. Was gently peeled from the substrate. The grown cells were seeded in 1.5 × 10 ⁷ cells in the above cell culture container, and centrifuged at 50 G for 12 hours to form a cell laminate on the bottom surface of the Petri dish. Subsequently, when the cell culture container was inverted, the cell laminate moved onto the OPS film, and the cell laminate could be transferred by separating the OPS film from the lid.

(Example 2)
Instead of adhering the OPS film by soaking toluene in the end of the lid, it was carried out except that the OPS film was bonded using a biocompatible deacetic acid type adhesive RTV118 (made by Momentive Performance Materials). A cell culture vessel was prepared in the same manner as in Example 1. Adhesion was performed by leaving it to stand for 2 days or more in a room temperature environment.

  A cell laminate was produced using this cell culture container, and the cell laminate was able to be transferred while being placed on the OPS film.

(Comparative Examples 1 and 2)
As Comparative Example 1, instead of forming a through-hole in the Petri dish lid, the bottom surface of the Petri dish was cut into a circular shape with an ultrasonic cutter, and the OPS film similar to Example 1 was adhered to that portion by a solvent welding method with toluene To prepare a cell culture container. Further, as Comparative Example 2, a cell culture container was produced in the same manner as in Comparative Example 1, except that the OPS film was adhered using the same adhesive as in Example 2.

  An attempt was made to produce a cell laminate by centrifugation using the cell culture containers of Comparative Examples 1 and 2. After centrifugation, the culture solution leaked from the gap between the bottom surface of the Petri dish and the OPS film, which was sufficient on the OPS film. No cell stack was formed.

(Comparative Example 3)
A film cut out in a circle along the shape of the bottom surface was laid on the bottom surface of the Petri dish to obtain a cell culture container of Comparative Example 3. An attempt was made to produce a cell laminate by centrifugation using this cell culture vessel.

  As a result, when the underlying film was smaller in size than the bottom surface of the Petri dish, cells were retained between the film and the bottom surface of the dish, and a cell laminate could not be produced. In addition, when the size of the film is the same as that of the bottom surface, it is not suitable because it becomes difficult to peel the film from the bottom surface of the dish after forming the cell laminate.

(Comparative Example 4)
As Comparative Example 4, a normal Petri dish that was not particularly processed was used, and a cell laminate was formed on the bottom of the dish by centrifugation, and the cell laminate was transferred using a film to transfer the cell laminate. We examined whether it was possible.

  As a result, although the cell laminate was formed, it was difficult to capture the cell laminate that was floating when rinsing with the film, which took time. Moreover, the phenomenon which depends on scooping a cell laminated body or the phenomenon where a cell laminated body is destroyed was seen.

(Weight measurement of cell stack)
In order to confirm that the present invention is useful in forming an appropriate tissue of a cell laminate, the following experiment was conducted.

  First, a cell culture container was prepared by the procedure of Example 1 and Comparative Example 1 using an OPS film whose weight was measured in advance, and a cell laminate was formed by applying centrifugal force under the same conditions as in Example 1. Subsequently, in the cell culture container according to Example 1, after centrifuging, the cell culture container was inverted, the culture solution was sucked out by a pipette, the OPS film was separated from the Petri dish lid, and the cell laminate was then removed from the OPS film. It collect | recovered in the state mounted on the top. In the cell culture container according to Comparative Example 1, the culture solution remaining after centrifugation was sucked out with a pipette, the OPS film was separated from the bottom surface of the Petri dish, and the cell stack was collected in a state of being placed on the OPS film. The total weight of each cell laminate and OPS film was measured, and the difference between the weight of the OPS film measured in advance was calculated to determine the weight of the cell laminate alone.

  As a result, when the cell culture container of Example 1 was used, a sufficient amount of the cell laminate was recovered, and the difference in weight was 0.5 g. On the other hand, when the cell culture container of Comparative Example 1 was used, the culture solution leaked from the gap between the bottom surface of the Petri dish and the OPS film by centrifugation, and almost no cell tissue was found on the OPS film. The difference in weight was 0.002 g. From the above results, it was revealed that the cell culture container of the present invention is useful for forming a sufficient amount of cell tissue as a cell laminate.

(Change in cell viability by film type)
In order to examine whether there is a difference in the viability of the cultured cells depending on the type of resin film adhered to the lid of the Petri dish, the following experiment was conducted.

As resin films covering the through-holes formed in the lid of the Petri dish, PET films (Asahi Kasei Chemicals Corp .; oxygen permeability 14.1 cc / m ² · day · atm), OPS films (Asahi Kasei Chemicals Corp .; oxygen permeation), respectively. Degree 50.3 cc / m ² · day · atm), polypropylene film (pyrene film-OT; manufactured by Toyobo; oxygen permeability 15 cc / m ² · day · atm), polyethylene film (IMX film; manufactured by J-Film; A cell culture vessel was prepared in the same manner as in Example 1 using a high oxygen permeability. The resin film and the Petri dish lid were bonded together with an adhesive. Using this cell culture vessel, a cell laminate was formed by centrifugation. Moreover, the cell laminated body was formed by centrifugation using a normal petri dish as a comparison object. In a normal petri dish, the oxygen permeability of the lid can be regarded as 0 cc / m ² · day · atm. The oxygen permeability of the resin film was measured in an atmosphere at a temperature of 23 ° C. and a relative humidity of 85% using a Mocon oxygen permeability meter (OX-TRAN 2/20).

  The prepared cell stack is washed with PBS, and then 1 μg / ml of Calcein-AM (Dojin Chemical Co., Ltd.) that stains living cells, 1 μg / ml of Propium Iodide (PI; Dojindo Co., Ltd.) that stains dead cells, and Hoechst 33342 (1/1000 dilution; Invitrogen) that stains cell nuclei was immersed in a reaction solution dissolved in PBS and reacted at 37 ° C. for 15 minutes. Thereafter, the cells were placed on a slide glass and sealed with glycerol and a cover glass, and then the state of cell survival was observed with a confocal microscope.

  The results are shown in Table 1. In Table 1, ◯ means that the number of cells is almost the same as that of the control (“dish”), Δ is slightly inferior, and X means that there are many dead cells. From this result, it was found that there was a difference in cell viability after centrifugation depending on the type of resin film. And the polyethylene film with high oxygen permeability has many dead cells and is unsuitable. Regarding the cell viability in Table 1, the approximate cell viability is determined by calculating the area ratio of Calcein-AM positive cells and PI positive cells by NIH ImageJ based on the acquired confocal microscope images. It was.

(Influence by cell type)
In order to examine whether or not the state of the obtained cell stack changes depending on the cell type, the following experiment was performed.

Cell stacks were prepared and collected using the cell culture vessel of Example 1 using mouse fibroblast CCL163 cells and rat chondrocyte MK442 (Takara Bio Inc.). The number of seeded cells was 1.5 × 10 ⁷ in both cases, and a PET film was used as a resin film to be adhered to the lid of the Petri dish, and was bonded together with an adhesive.

  As a result of the experiment, a cell laminate was obtained in the same manner for the above two types of cells, and it was excellent in handling properties, and could be transferred without breaking or deforming the cell laminate.

  Next, the same examination was performed using MDCK cells having strong adhesion. Three cell culture containers are Petri dishes (manufactured by BD), polyethylene glycol (PEG) coated dishes, and HydroCell (manufactured by Cell Seed) 3.5 cm dishes, and through holes are formed in these lids. The through hole was covered with a PET film. The PEG coating dish was manufactured by coating the surface with PEG400 (manufactured by Sanyo Kasei Co., Ltd.) according to the method described in Japanese Patent No. 5252636. HydroCell is a dish that has been surface-treated to make it non-cell-adhesive by coating the surface with an acrylamide-based substance.

First, in order to detach the cells grown in the medium, the reaction was performed at 37 ° C. for 10 minutes with a 0.25% trypsin solution. The cell seeding amount for the cell culture container is 1.5 × 10 ⁷ cells, and the cell laminate is formed by centrifuging at 50 G for 12 hours, and the cell culture container is placed on the PET film by inverting the cell culture container. We examined whether body recovery was performed. The results are shown in Table 2. In Table 2, “O” represents that the cell laminate was properly collected, and “X” represents that the collection was difficult.

  As shown in Table 2, the bottom surface of the Petri dish was insufficient in cell non-adhesiveness with respect to MDCK cells, and the cell laminate adhered to the bottom surface of the Petri dish, and it was difficult to recover the PET film. . Naturally, the handling property of the cell laminate was also poor. On the other hand, in the two dishes whose surfaces were coated with a hydrophilic polymer, the formed cell laminate was floating after centrifugation, and it was easy to recover to a PET film.

DESCRIPTION OF SYMBOLS 1 Cell culture container 10 Bottom part 101 Inner surface 11 Container member 20 Upper part 21 Lid member 210 Through-hole A Cell laminated body

Claims (4)

A bottom portion having an inner surface that is non-cell-adhesive or capable of changing to cell-non-adhesion, and an upper portion facing the bottom portion, the upper portion being 200 cc / m ² · day · atm or less A cell culture vessel having an oxygen permeability of 1 and configured to be separable. The cell culture container according to claim 1, wherein the oxygen permeability is 15 cc / m ² · day · atm or less.   The cell culture vessel according to claim 2, wherein the upper part is a polyethylene terephthalate film or a laminated film containing the polyethylene terephthalate film. A bottom portion having an inner surface that is non-cell-adhesive or capable of changing to cell-non-adhesion, and an upper portion facing the bottom portion, the upper portion being 200 cc / m ² · day · atm or less A cell addition step of placing a culture solution containing cells in a cell culture vessel that is configured to be separable with oxygen permeability of:
A cell culturing step of performing cell culture under a condition in which cell-cell adhesion is formed while acting centrifugal force toward the inner surface of the bottom portion on the cells, and bonding the cells to form a cell stack,
Reversing the positions of the bottom and the top, and separating the resulting cell stack from the inner surface and placing it on the top;
A separation step of separating the upper part together with the placed cell stack;
A method for producing a cell laminate comprising:

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