JP6690119B2 - Cell culture container and method for producing cell stack - Google Patents

Description

  The present invention relates to a cell culture container and a method for producing a cell stack 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 the detachment of cell sheets, a technology has been developed to accelerate the detachment of cells by providing a layer of temperature-responsive polymer on the cell adhesion surface, and it is possible to collect cell sheets containing almost no foreign matter. 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, it is necessary to stack a plurality of cell sheets in order to form a multilayer. Since cell sheets are extremely thin and difficult to handle, it is difficult and time-consuming to stack a plurality of cells.

  As a method for easily producing a multi-layered cell sheet (cell stack), (Patent Document 1) discloses that cells are non-adhesive or can be changed to cell non-adhesive. Under the condition that cell-cell adhesion is formed, while adding a culture solution containing cells to the culture vessel having a bottom surface and applying centrifugal force to the cells added to the culture vessel toward the inner bottom surface. Disclosed is a method for producing a tissue, which comprises a cell culture step of performing cell culture and adhering cells to form a tissue, and a peeling step of peeling and collecting the tissue obtained in the cell culture step from the inner bottom surface. ing.

  In this method, it is possible to easily control the number of cell stacks of a tissue and produce a biological tissue in a shorter time, but since an enzyme is used to exfoliate cells grown in a culture solution. However, there was room for improvement in handleability because the intercellular bond in the produced tissue was weak and the strength of the tissue was weak. Therefore, it has been desired to develop a container that can be easily transferred without destroying the tissue of the cell stack produced by using the centrifugal force, and a method for producing the cell stack using the container.

Japanese Patent No. 5407343

  In view of the above conventional circumstances, the present invention provides a cell culture container that can be easily transferred without disrupting the shape of a cell stack produced using centrifugal force, and a method for producing a cell stack using the same. The purpose is to

  In order to solve the above problems, as a result of intensive studies by the present inventors, the upper portion of the cell culture container has a predetermined oxygen permeability and is configured to be separable, and the cell stack is placed on the upper portion. The inventors have found that the handling property of the cell stack body is improved by transporting the cells as described above and completed the invention. That is, the gist of the present invention is as follows.

(1) It has a bottom having an inner surface that is non-cell-adhesive or capable of changing into a cell-non-adhesive, and an upper part that faces the bottom, and the upper part 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) above, which has an oxygen permeability of 15 cc / m ² · day · atm or less.
(3) The cell culture container according to (2) above, wherein the upper part is a polyethylene terephthalate film or a laminated film containing the polyethylene terephthalate film.
(4) It has a bottom part having an inner surface that is non-cell-adhesive or capable of changing into a cell-non-adhesive property, and an upper part facing the bottom part, wherein the upper part is 200 cc / m ² · day. A cell addition step of adding a culture solution containing cells to a cell culture container that has an oxygen permeability of atm or less and is configured to be separable;
While applying a centrifugal force to the cells toward the inner surface of the bottom, cell culture is performed under conditions where cell-cell adhesion is formed, and a cell culture step of forming a cell stack by adhering cells.
A placing step of reversing the positions of the bottom portion and the upper portion, peeling the obtained cell stack from the inner surface and placing the cell laminated body on the upper portion,
A separation step of separating the upper part together with the placed cell stack,
A method for producing a cell stack having:

  According to the present invention, the cell stack produced by utilizing the centrifugal force can be easily recovered and transferred without causing damage or deformation.

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

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

  The method for producing a cell laminate of the present invention has a bottom portion having an inner surface that is non-adhesive to cells or capable of changing into non-adhesive cells, and an upper portion facing the bottom portion, and the upper portion is While adding a culture solution containing cells to a cell culture container that has a predetermined oxygen permeability and is separable, while applying a centrifugal force to the cells toward the inner surface of the bottom, , The cell culture step of culturing cells under the condition that cell-cell adhesion is formed and adhering cells to form a cell stack, and inverting the positions of the bottom and top, And a separation step of separating the upper part together with the mounted cell stack from each other.

1. Cell Addition Step First, the configuration of a cell culture vessel for containing a culture solution containing cells in the cell addition step will be described. FIG. 1 shows one embodiment of the cell culture container. The cell culture container 1 of 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 portion 10 is configured as a part of a petri dish-shaped container member 11 having a side wall. The upper portion 20 is stacked on the upper surface of the lid member 21 that is combined with the container member 11. A through hole 210 is formed in the center of the lid member 21, the through hole 210 is covered by the upper portion 20, and the upper portion 20 is exposed to the internal space of the cell culture container. It should be noted that the present invention is not limited to the embodiment of FIG. 1, and for example, the upper part 20 also serves as the lid member 21, and the film-like or plate-like upper part 20 is directly laminated on the upper surface of the dish-like container member 11 to form the container member 11. It may be configured to cover the opening.

  The planar view areas of the container member 11, the lid member 21, and the upper portion 20 are not particularly limited, but are preferably larger than the cell stack body to be produced. 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, but it is a circular shape from the viewpoint of easiness of manufacturing and handling. Preferably there is. Furthermore, it is preferable that the container member 11, the lid member 21, and the upper portion 20 constituting the cell culture container 1 be sterilized in advance in order to handle cells. The sterilization method is not particularly limited, 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 may be in the form of a film or a plate, but in consideration of handleability when the cell stack produced on the upper part 20 is transferred and used for transplantation work, etc., it has flexibility. It is preferable that the resin film has, and it is preferable that the resin film is transparent for visually recognizing cells. The thickness of the upper part 20 is not particularly limited as long as it has a predetermined oxygen permeability, but if it is too thin, the handling property at the time of transferring together with the cell stack is deteriorated, and the centrifugal force is reduced. When it is added, it is likely to be deformed. On the contrary, if it is too thick, it takes time and effort to separate it from the lid member 21. Specifically, when the upper portion 20 is made of a resin film, the thickness is preferably 30 μm to 100 μm, particularly preferably 40 μm to 80 μm, although it varies depending on the type of resin.

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. High oxygen permeability increases the oxygen concentration in the culture medium compared to when using a normal culture dish, which may lead to cytotoxicity and reduce viability (Forsyth et al., Aging Cell). , Vol. 2, pp. 235-243, 2003). Further, particularly when the cell stack is formed by utilizing the centrifugal force, maintaining the pH of the culture solution by controlling the CO ₂ concentration is important for improving the cell viability. When 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 not, the CO ₂ concentration of the culture solution becomes almost the same as the outside air, and Cell death may occur due to a decrease in CO ₂ concentration and a shift of the culture medium to be alkaline. Therefore, by controlling the oxygen permeability of the upper part 20 to be 200 cc / m ² · day · atm or less, it becomes 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, a good cell laminate can be produced without decreasing the cell viability.

  The material of the upper portion 20 is not particularly limited as long as it satisfies the above oxygen permeability conditions. 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. A polypropylene (PP) or polystyrene (PS) film is also applicable because it has a relatively high oxygen permeability but is within the above range.

When a resin film is used as the upper portion 20, the resin film may be a single layer, but a laminated film obtained by laminating a plurality of resin films may be used. The polyethylene (PE) film may have an oxygen permeability of more than 200 cc / m ² · day · atm when used as a single layer, but it is used as a laminated film to increase the barrier property by increasing the thickness. , The upper part 20 can be used. A polyethylene terephthalate film or a laminated film containing a polyethylene terephthalate film is preferably used as the upper portion 20.

  The method for adhering the upper portion 20 and the lid member 21 is not particularly limited, and various methods such as an adhering method using a solvent, an adhering method using an adhesive, and heat sealing can be appropriately adopted. When using an adhesive, it is desirable that the adhesive be composed of components having biocompatibility. In addition, the adhesive force is not particularly limited as long as it can separate the upper portion 20 after forming the cell stack. Specifically, it is desirable to be in the range of 3N to 6N / 15 mm according to JIS Z0238.

  The cell culture container 1 used in the present invention may be configured such that at least the inner surface 101 of the bottom portion 10 is cell non-adhesive or can be changed to cell non-adhesive, and in particular, Of the inner surfaces of the container member 11 that contains the culture medium containing cells, all 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-adhesive or non-cell-adhesive. It is preferable that it is possible to change the sex.

  The cells used in the present invention are not particularly limited as long as they are adhesive cells. Examples of such cells include hepatocytes that are parenchymal cells of the liver, 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 gland cells, pericytes, muscle cells such as smooth muscle cells and cardiomyocytes, renal cells , Pancreatic islets of Langerhans, nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, and bone cells. These cells may be primary cells directly collected from a tissue or organ, or may be those obtained by subculturing them for several generations. Further, these cells are undifferentiated embryonic stem cells, pluripotent stem cells such as pluripotent mesenchymal stem cells, unipotent stem cells such as unipotential vascular endothelial progenitor cells, etc. It may be any of the cells. The cells may be cultivated in a single kind or may be co-cultured in two or more kinds.

  These cells are preliminarily cultivated by a usual method, the culture is treated with trypsin treatment and the like, and then stored in the cell culture container 1 in a state of being suspended in the culture medium. 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. The basal medium as described in "Technology of Tissue Culture, Third Edition", edited by The Tissue Culture Society of Japan, p. 581, can be used. Furthermore, serum (fetal bovine serum, etc.), various growth factors, antibiotics, amino acids, etc. may be added to the basal medium. Further, a commercially available serum-free medium such as Gibco serum-free medium (Invitrogen) can be used. Considering the clinical application of the finally obtained cell stack, it is preferable to use a medium containing no animal-derived component.

In the present invention, “non-adhesive to cells” refers to non-adhesiveness to cells to be cultured, and non-adhesiveness to cells refers to a property in which cells hardly adhere. The cell non-adhesiveness is determined by whether or not cell adhesion and spreading hardly occur due to the chemical and physical properties of the surface. Specifically, when the cell adhesion extension rate in actual cell culture is used as an index, the case where the cell adhesion extension rate is less than 60% is defined as cell non-adhesion. 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 spreading rate is determined by seeding cells to be cultivated at a seeding density in the range of 4000 cells / cm ² or more and less than 30,000 cells / cm ^{2 on the} surface to be measured, and incubating at 37 ° C., 5% CO ₂ concentration in an incubator. It is defined as the ratio of cells that have been adhered and expanded at the time of culturing for 14.5 hours ({(number of adhered cells) / (number of seeded cells)} × 100 (%)). Therefore, depending on the type of cells, a dish for suspension culture such as Petri dish can be used as it is. Alternatively, a surface treatment that makes cells non-adhesive can be appropriately applied. In the case of cells with strong adhesiveness (for example, canine renal 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, which may make recovery difficult. It is preferable to apply a surface treatment to form a cell-nonadhesive surface.

  Examples of the cell non-adhesive surface include a hydrophilic surface, specifically, a surface having a static water contact angle of 20 ° C. of 45 ° or less. However, a surface having enhanced cell adhesiveness due to hydrophilic treatment such as plasma treatment or corona treatment is not preferable. The properties of the surface can be examined by measuring the O component on the surface by means such as elemental analysis and estimating the amount of hydroxyl groups on the surface. The cell-non-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 portion 10. Alternatively, the bottom 10 itself may be made of a hydrophilic material.

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

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

  Specific water-soluble polymer materials include polyalkylene glycol and its derivatives, polyacrylic acid and its derivatives, polymethacrylic acid and its derivatives, polyacrylamide and its derivatives, polyvinyl alcohol and its derivatives, zwitterionic polymers. , Polysaccharides and the like. The molecular shape may be linear, branched or dendrimer. More specifically, polyethylene glycol, a copolymer of polyethylene glycol and polypropylene glycol, poly (N-isopropylacrylamide), poly (N-vinyl-2-pyrrolidone), poly (2-hydroxyethylmethacrylate), poly (methacryloyl). Oxyethylphosphorylcholine), methacryloyloxyethylphosphorylcholine, a copolymer of 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 their derivatives, acrylic acid oligomers and their derivatives, methacrylic acid oligomers and their derivatives, acrylamide oligomers and their derivatives, saponified vinyl acetate oligomers and their 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. You can 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 thereof 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 0.8 nm to 500 μm, more preferably 0.8 nm to 100 μm, further preferably 1 nm to 10 μm, most preferably 1.5 nm to 1 μm. If the average thickness is 0.8 nm or more, the inner surface 101 of the bottom portion 10 is unlikely to be affected, which is preferable. Further, if the average thickness is 500 μm or less, coating is relatively easy.

  As a method for forming the hydrophilic film on the inner surface 101 of the bottom portion 10, a method of directly adsorbing the main raw material (hydrophilic organic compound etc.) of the hydrophilic film to the inner surface 101, or a method of directly coating the inner surface 101 with the hydrophilic organic compound etc. Method, method of applying cross-linking treatment after coating the inner surface 101 with a hydrophilic organic compound, method of forming hydrophilic film in a multi-step manner to improve adhesion to the inner surface 101, and improvement of adhesion with the bottom portion 10. For that purpose, a method of forming a base layer on the inner surface 101 and then coating a hydrophilic organic compound or the like, a method of forming a polymerization initiation point on the inner surface 101 and then polymerizing a hydrophilic polymer brush, and the like can be mentioned.

  Among the above-mentioned film forming methods, particularly preferable methods include a method of forming a hydrophilic film in a multi-step manner, and a method of forming an underlayer on the inner surface 101 to improve the adhesion with the bottom portion 10 and then forming a hydrophilic organic layer. A method of coating a compound or the like can be mentioned. This is because when these methods are used, it is easy to enhance the adhesion of the hydrophilic organic compound or the like to the bottom portion 10. The term "bonding layer" is used herein. In the case of forming a film of a hydrophilic organic compound or the like in a multi-step manner, the binding layer means a layer existing between the hydrophilic film layer on the outermost surface and the bottom portion 10, and the inner layer 101 is provided with a base layer. When a hydrophilic film layer is formed on the underlayer, it means the underlayer. The binding layer is preferably a layer containing a material having a binding portion (linker). Examples of the combination 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, either may be a linker. In these methods, a bonding layer is formed on the inner surface 101 by a material having a linker before coating with a hydrophilic material. The density of the material in the tie layer is an important factor defining the bond strength. 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, if the water contact angle of the inner surface 101 to which the 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 portion 10 of the cell culture container 1 used in the present invention is preferably non-cell-adhesive from the viewpoint of easy peeling of the cell stack, but as another form, cell-adhesiveness during cell culture. However, it may be a surface that can change into cell non-adhesiveness upon peeling. Such a surface has at least one stimuli-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 via a covalent bond. Can be formed by. The stimuli-responsive polymer is preferably a temperature-responsive polymer, but is not limited thereto.

  The temperature-responsive polymer that can be preferably used in the present invention is hydrophobic at the cell culture temperature (usually about 37 ° C.) and hydrophilic at the temperature at which the formed cell stack is recovered. The temperature at which the temperature-responsive polymer changes from hydrophobic to hydrophilic (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 than that. By including such a temperature-responsive polymer component, a cell scaffold (cell adhesion surface) is sufficiently secured during cell culture, so that cell culture can be efficiently performed. On the other hand, at the time of collecting the cell stack, by changing the hydrophobic portion to hydrophilic and separating the cell stack from the bottom portion 10, the collection of the cell stack can be further facilitated. 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 dissolution temperature of such a temperature responsive polymer is particularly called the lower limit critical dissolution temperature.

  The temperature-responsive polymer that can be preferably used in the present invention is specifically a polymer having a lower critical solution temperature T of 0 ° C to 80 ° C, preferably 0 ° C to 50 ° C. When T exceeds 80 ° C, cells may be killed, which is not preferable. Further, if T is lower than 0 ° C., the cell growth rate is generally extremely decreased or the cells are killed, which is not preferable. Such suitable polymers include acrylic or methacrylic polymers. Specifically, for example, poly-N-isopropylacrylamide (T = 32 ° C), poly-Nn-propylacrylamide (T = 21 ° C), poly-Nn-propylmethacrylamide (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-diethyl acrylamide (T = 32 degreeC) etc. are mentioned. Other polymers include, for example, poly-N-ethylacrylamide, poly-N-isopropylmethacrylamide, poly-N-cyclopropylacrylamide, poly-N-cyclopropylmethacrylamide, poly-N-acryloylpyrrolidine, poly-N. -Acryloyl piperidine, polymethyl vinyl ether, methyl cellulose, ethyl cellulose, alkyl-substituted cellulose derivatives such as hydroxypropyl cellulose, polyalkylene oxide block copolymers represented by block copolymers of polypolypropylene oxide and polyethylene oxide, and poly Examples thereof include alkylene oxide block copolymers.

  Examples of the monomer for forming these polymers include monomers in which a homopolymer of the monomer has T = 0 ° C. to 80 ° C. and which can be polymerized by irradiation with radiation. 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 one species 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 included in the present invention. Further, when T needs to be adjusted depending on the type of proliferating cells, a monomer other than the above may be further added for copolymerization. Further, a graft or block copolymer of the above polymer with another polymer, or a mixture of a temperature responsive polymer or the like with another polymer may be used. Further, 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 produced can be appropriately selected.

2. Cell culture step In the cell culture step, the cells in the culture medium contained in the cell culture container 1 are subjected to a centrifugal force in the direction of the inner surface 101 of the bottom portion 10 while the cell culture is performed under the condition that cell-cell adhesion is formed. Is performed, and the cells are adhered to each other to form a cell stack. Through this step, as shown in FIG. 2, the cell stack A is formed on the inner surface 101 of the bottom portion 10.

  The magnitude of the centrifugal force can be appropriately selected within a range in which the cell stack can be formed without adversely affecting the cell function. For example, the range of 2G to 1440G is preferable, and the range of 2G to 720G is more preferable. A centrifugal force can be applied by placing the cell culture container 1 containing the cell-containing culture solution in a centrifuge and performing a centrifugation operation.

  “Conditions for forming cell-cell adhesion” refer to conditions under which cells are activated and cells can 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%, and the culture time is 0. 5 hours to 24 hours are preferable. By applying the centrifugal force, the culture time can be shortened and the damage to the cells can be reduced. The culturing time can be shortened by separately preparing and adding the extracellular matrix, and the culturing time can be 0.5 to 3 hours. When the extracellular matrix is not added, the culture time can be set to 1 hour to 24 hours.

  In the cell culture step, it is sufficient that cell-cell adhesion is formed, and it is not essential to increase the cell number 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 grow cells in the cell culture step, a cell stack can be obtained in a relatively short time. Further, the thickness and shape of the tissue of the cell stack can be adjusted freely.

  Further, by utilizing the centrifugal force, it becomes possible to obtain a cell stack in which cells are densified. The cell stack having a high density of cells can be suitably used for transplantation.

3. Mounting Step In the mounting step, after the centrifugal operation is completed, as shown in FIG. 3, the positions of the bottom portion 10 and the upper portion 20 are inverted, and the obtained cell stack A is peeled from the inner surface 101 of the bottom portion 10 and the upper portion 20 is removed. This is the step of placing on. If the inner surface 101 is a cell-non-adhesive surface, the peeling operation is easy. If necessary, a tool such as tweezers may be used supplementarily, or the cell stack A may be moved to the upper side 20 by the action of gravity. It can be done. In addition, when the inner surface 101 is a surface such as a stimuli-responsive polymer that can be changed to be non-cell adhesive, an environment (for example, a temperature equal to or lower than the lower limit critical temperature) that makes the cell non-adhesive. ), The peeling operation is performed.

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

  Next, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the scope of the present invention is not limited to these Examples.

(Example 1)
Using a 3.5 cm size Petri dish (manufactured by BD), a circular through hole having an appropriate size (about 25 to 30 mm in diameter) was formed on the lid of this Petri dish with an ultrasonic cutter, and then the lid The edges were soaked with toluene to allow the film to adhere. Next, a biaxially stretched polystyrene film (OPS film; manufactured by Asahi Kasei Chemicals Corp.) was cut into a circle of 35 mm size, adhered to the lid to cover the through hole, and 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), and after reaching confluence, cells were reacted with 0.01% trypsin for 10 minutes at 37 ° C. Was gently peeled from the substrate. The grown cells were seeded in the above cell culture vessel at 1.5 × 10 ⁷ cells and centrifuged at 50 G for 12 hours to form a cell stack on the bottom surface of the Petri dish. Subsequently, when the cell culture vessel was inverted, the cell stack moved to the OPS film, and the cell stack could be transferred by separating the OPS film from the lid.

(Example 2)
Except that the OPS film was adhered using a deacetic acid type adhesive RTV118 (manufactured by Momentive Performance Materials) having biocompatibility instead of allowing the end of the lid to be impregnated with toluene and adhering the OPS film. A cell culture vessel was prepared in the same manner as in Example 1. The adhesion was performed by leaving it in a room temperature environment for 2 days or more.

  A cell stack was prepared using this cell culture container, and the cell stack could 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 lid of the Petri dish, the bottom surface of the Petri dish was cut out in a circular shape with an ultrasonic cutter, and the OPS film similar to that of Example 1 was adhered to that portion by a solvent welding method using toluene. Then, a cell culture container was produced. Further, as Comparative Example 2, a cell culture container was produced in the same manner as Comparative Example 1 except that the OPS film was adhered using the same adhesive as in Example 2.

  Attempts were made to produce a cell stack by centrifugation using the cell culture vessels of Comparative Examples 1 and 2, and after the centrifugation, the culture solution leaked from the gap between the bottom of the Petri dish and the OPS film, which was sufficient on the OPS film. No cell stacks were formed.

(Comparative example 3)
A film cut into 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 stack by centrifugation using this cell culture container.

  As a result, when the underlying film had a smaller size than the bottom surface of the Petri dish, cells were retained between the film and the bottom surface of the dish, so that a cell stack could not be prepared. Further, when the size of the film was the same as that of the bottom surface, it was not suitable because it was difficult to peel off the film from the bottom surface of the dish after forming the cell stack.

(Comparative example 4)
As Comparative Example 4, an ordinary unprocessed Petri dish was used, a cell stack was formed on the bottom of this dish by centrifugation, and then the cell stack was scooped using a film to transfer the cell stack. I examined whether I could do it.

  As a result, although the cell stack was formed, it was difficult and time-consuming to catch the floating cell stack when scooping with the film. In addition, there was a phenomenon in which the cell stack was damaged by scooping or the cell stack was destroyed.

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

  First, a cell culture vessel was prepared by using the OPS film whose weight was measured in advance according to the procedure of Example 1 and Comparative Example 1 described above, and a centrifugal force was applied under the same conditions as in Example 1 to form a cell stack. Subsequently, in the cell culture container according to Example 1, after centrifuging, the cell culture container was inverted, the culture solution was sucked out with a pipette, and then the OPS film was separated from the lid of the Petri dish to give the cell stack to the OPS film. It was collected while it was placed on 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 the OPS film was measured, and the difference between the weight of the OPS film and the previously measured weight was calculated to obtain 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 stack was recovered, and the difference in weight was 0.5 g. On the other hand, when the cell culture vessel of Comparative Example 1 was used, the culture solution leaked from the gap between the bottom of the Petri dish and the OPS film by centrifugation, and almost no cell tissue was found on the OPS film. The weight difference was 0.002 g. From the above results, it became clear that the cell culture container of the present invention is useful for forming a sufficient amount of cell tissue as a cell stack.

(Change in cell viability depending on film type)
The following experiment was performed to examine whether the viability of the cells to be cultured varies depending on the type of resin film attached to the lid of the Petri dish.

As a resin film covering the through holes formed in the lid of the Petri dish, a PET film (manufactured by Asahi Kasei Chemicals; oxygen permeability 14.1 cc / m ² · day · atm) and an OPS film (manufactured by Asahi Kasei Chemicals; oxygen permeation) 50.3 cc / m ² · day · atm), polypropylene film (Pyrene film-OT; manufactured by Toyobo Co., Ltd .; oxygen permeability 15 cc / m ² · day · atm), polyethylene film (IMX film; manufactured by J-Film Co., Ltd.); A cell culture vessel was produced in the same manner as in Example 1 using the oxygen permeability (high). The resin film and the Petri dish lid were attached by an adhesive. Using this cell culture vessel, a cell stack was formed by centrifugation. As a comparative object, a normal petri dish was used to form a cell stack by centrifugation. 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 using an oxygen permeability meter (OX-TRAN 2/20) manufactured by Mocon Co., Ltd. in an atmosphere of a temperature of 23 ° C. and a relative humidity of 85%.

  After washing the prepared cell stack with PBS, 1 μg / ml Calcein-AM (Dojindo Co., Ltd.) that stains live cells, 1 μg / ml Propium Iodide (PI; Dojindo Corp.) 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. Then, the cells were placed on a slide glass, sealed with glycerol and a cover glass, and then the state of cell survival was observed by a confocal microscope.

  The results are shown in Table 1. In Table 1, ◯ means that the number of cells was about the same as that of the control (“dish”), Δ was slightly inferior, and × means that there were many dead cells. From this result, it was found that the cell viability after centrifugation was different depending on the type of resin film. The polyethylene film having a high oxygen permeability was not suitable because it had many dead cells. Regarding the cell viability in Table 1, the approximate cell viability was determined by calculating the area ratio of each of Calcein-AM positive cells and PI positive cells by NIH ImageJ based on the acquired confocal microscope image. It was

(Influence by cell type)
The following experiment was conducted to examine whether the state of the obtained cell stack changes depending on the cell type.

Using CCL163 cells of mouse fibroblasts and MK442 of rat chondrocytes (Takara Bio Inc.), a cell stack was prepared and collected in the cell culture container of Example 1. The number of seeded cells was both 1.5 × 10 ⁷ , and a PET film was used as the resin film adhered to the lid of the Petri dish, and the PET film was adhered with an adhesive.

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

  Next, the same examination was performed using MDCK cells having strong adhesiveness. As a cell culture container, Petri dish (manufactured by BD), polyethylene glycol (PEG) coated dish, and HydroCell (manufactured by Cell Seed) of 3.5 cm dish were used as container members, and through holes were formed in these lids. The thing which covered the through-hole with the PET film was used. The PEG coated dish was produced by coating the surface with PEG400 (manufactured by Sanyo Kasei Co., Ltd.) according to the method described in Japanese Patent No. 5252036. In addition, HydroCell is a dish that has been subjected to a surface treatment that renders cells non-adhesive by surface-coating an acrylamide substance.

First, in order to detach the cells grown in the medium, they were reacted with a 0.25% trypsin solution at 37 ° C. for 10 minutes. The cell seeding amount in the cell culture container was set to 1.5 × 10 ⁷ , and the cell stack was formed by centrifuging at 50 G for 12 hours, and the cell culture container was inverted to stack the cells on the PET film. We examined whether body recovery would take place. The results are shown in Table 2. In Table 2, ◯ represents that the cell stack was properly collected, and x represents that the collection was difficult.

  As shown in Table 2, the bottom surface of the Petri dish had an insufficient degree of cell non-adhesiveness to MDCK cells, and the cell stack adhered to the bottom surface of the Petri dish and was difficult to collect on a PET film. . Naturally, the handling property of the cell stack was also poor. On the other hand, in the two dishes having the surface coating with the hydrophilic polymer, the formed cell laminate floated after centrifugation, and it was easy to collect it on the PET film.

1 Cell Culture Container 10 Bottom 101 Inner Surface 11 Container Member 20 Upper Part 21 Lid Member 210 Through Hole A Cell Stack

Claims (1)

A bottom part having an inner surface that is non-cell-adhesive or capable of changing into a non-cell-adhesive form; a top film facing the bottom part; and a lid member that detachably supports the top film. A cell adding step in which a culture solution containing cells is placed in a cell culture container in which the upper film has an oxygen permeability of 200 cc / m ² · day · atm or less,
While applying a centrifugal force to the cells toward the inner surface of the bottom, cell culture is performed under conditions where cell-cell adhesion is formed, and a cell culture step of forming a cell stack by adhering cells.
A placing step of reversing the positions of the bottom portion and the upper film, placing the obtained cell laminate on the upper film together with peeling from the inner surface,
A separation step of separating the upper film from the lid member together with the placed cell stack,
A method for producing a cell stack having:

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