JP6835266B2 - Method for preparing cell culture vessel and cell laminate - Google Patents

Description

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

Seeding cells on culture supports to form cell sheets is commonly practiced. For the purpose of facilitating the peeling of cell sheets, a technology has also been developed in which a layer of a temperature-responsive polymer compound is provided on the cell adhesion surface to promote cell peeling, and it is possible to recover cell sheets containing almost no foreign substances. Is. However, the cell sheet formed by this method is usually composed of a single layer or three or less cell layers. Therefore, it is necessary to stack a plurality of cell sheets in order to perform multi-layering. Since the cell sheet is extremely thin and difficult to handle, it is not easy and time-consuming to stack a plurality of cell sheets.

As a method for easily producing a multi-layered cell sheet (cell laminate), (Patent Document 1) states that the cells are non-adhesive or can be changed to non-cell adhesive. Under the condition that cell-cell adhesion is formed while applying a cell addition step of adding a culture solution containing cells to a culture vessel having a bottom surface and applying a centrifugal force toward the inner bottom surface to the cells added to the culture vessel. 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 produce a living tissue in a shorter time while easily controlling the number of cells stacked in the tissue, but since an enzyme is used to exfoliate the cells grown in the culture medium. There was room for improvement in handleability due to the weak cell-cell connection in the prepared tissue and the weak tissue strength. Therefore, it has been desired to develop a container that can be easily transferred without destroying the tissue of the cell laminate prepared by using centrifugal force, and a method for preparing the cell laminate using the container.

Japanese Patent No. 5407343

In view of the above-mentioned conventional situation, the present invention provides a cell culture container capable of easily transferring a cell laminate prepared by using centrifugal force without losing its shape, and a method for preparing the cell laminate using the cell culture container. The purpose is.

As a result of diligent research conducted by the present inventors in order to solve the above problems, the upper part of the cell culture vessel is configured to have a predetermined oxygen permeability and is separable, and a cell laminate is placed on the upper part. The invention was completed by finding that the handleability of the cell laminate was improved by transferring the cells. 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 can be changed to non-cell-adhesive, and an upper portion facing the bottom, and the upper portion is 200 cc / m ² · day. -A cell culture container having an oxygen permeability of atm or less and being separable.
(2) The cell culture vessel according to (1) above, wherein the oxygen permeability is 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 having an inner surface which is non-cell-adhesive or can be changed to non-cell-adhesive, and an upper part facing the bottom, and the upper part is 200 cc / m ² · day. -A cell addition step of putting a culture solution containing cells into a cell culture container having an oxygen permeability of atm or less and being configured to be separable.
A cell culture step in which cells are cultured under conditions where cell-cell adhesion is formed while applying centrifugal force toward the inner surface of the bottom of the cells, and the cells are adhered to form a cell laminate.
A mounting step of reversing the positions of the bottom and the top, peeling the obtained cell laminate from the inner surface, and placing the cell laminate on the top.
A separation step of separating the upper part together with the placed cell laminate,
A method for producing a cell laminate having.

According to the present invention, a cell laminate prepared by utilizing centrifugal force can be easily collected and transferred without causing destruction, deformation or the like.

It is a side sectional view (a) and a plan view (b) which show one Embodiment of the cell culture container which concerns on this invention. It is a figure for demonstrating the cell culture process in the method for producing the cell laminate which concerns on this invention. It is a figure for demonstrating the placement process in the manufacturing method of the cell laminate which concerns on this invention. It is a figure for demonstrating the separation process in the manufacturing method of the cell laminate 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 having an inner surface that is non-cell-adhesive or can be changed to non-adherent cells, 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 container having a predetermined oxygen permeability and being configured to be separable, and applying a centrifugal force toward the inner surface of the bottom of the cells. , Cell culture is performed under the condition that cell-cell adhesion is formed, cells are adhered to form a cell laminate, and the positions of the bottom and top are reversed, and the obtained cell laminate is used as the bottom. It is characterized by having a placement step of peeling from the inner surface of the cell and placing it on the upper part, and a separation step of separating the upper part together with the placed cell laminate.

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

The plan-view area of the container member 11, the lid member 21, and the upper portion 20 is not particularly limited, but is preferably larger than the cell laminate to be produced. Further, the planar shapes of the container member 11, the lid member 21, and the upper portion 20 are not particularly limited and may be polygonal rather than circular, but are circular from the viewpoint of ease of manufacture and handling. It is preferable to have. Further, it is preferable that the container member 11, the lid member 21, and the upper portion 20 constituting the cell culture container 1 are 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 portion 20 may be in the form of a film or a plate, but in consideration of handleability when the cell laminate produced on the upper portion 20 is transferred in a placed state and used for transplantation work or the like, flexibility is provided. It is preferably a resin film having a resin film, and is preferably transparent so that cells can be visually recognized. The thickness of the upper portion 20 may be a thickness having a predetermined oxygen permeability and is not particularly limited, but if it is too thin, the handleability at the time of transfer with the cell laminate is lowered, and the centrifugal force is also applied. If it is too thick, it will take time and effort to separate it from the lid member 21, so that it is appropriately set in consideration of the balance between them. Specifically, when the upper portion 20 is made of a resin film, it is preferably in the range of 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 using a normal culture dish, which may cause cytotoxicity and thus reduce survival (Forsyth et al., Aging Cell). , Volume 2, pp. 235-243, 2003). Further, especially when a cell laminate is formed by utilizing centrifugal force, it is important to maintain the pH of the culture solution by controlling _{the CO 2 concentration in order to improve the cell viability.} If the oxygen permeability of the upper part 20 is high, there is no problem if the centrifuge is always supplied with _{CO 2, but if it is not, the CO 2} concentration of the culture solution becomes almost the same as the outside air, and the culture solution 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 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.} A good cell laminate can be produced without reducing the cell viability without using the same centrifuge.

The material of the upper portion 20 may satisfy the above-mentioned oxygen permeability conditions, and is not particularly limited. When a resin film is used as the upper portion 20, as a specific example, a film such as polyethylene terephthalate (PET), ethylene / vinyl alcohol copolymer resin (EVOH), polyvinylidene chloride, nylon, cellophane or the like is preferably used. Further, 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, or a laminated film in which a plurality of resin films are laminated may be used. When used as a single layer, the polyethylene (PE) film may have an oxygen permeability ^{of more than 200 cc / m 2} , day, atm, but by using it as a laminated film, the thickness is increased to enhance the barrier property. , Can be used as the upper 20. As the upper portion 20, a polyethylene terephthalate film or a laminated film containing a polyethylene terephthalate film is particularly preferably used.

The method of adhering the upper portion 20 and the lid member 21 is not particularly limited, and various methods such as an adhesive method using a solvent, an adhesive method, and a heat seal can be appropriately adopted. When an adhesive is used, it is desirable that the adhesive is composed of components having biocompatibility. Further, the adhesive strength is not particularly limited as long as the upper portion 20 can be separated after forming the cell laminate. Specifically, it is desirable that it is within the range of 3N to 6N / 15 mm according to JIS Z0238.

The cell culture vessel 1 used in the present invention may be configured such that at least the inner surface 101 of the bottom 10 is non-cell-adhesive or can be changed to non-cell-adhesive, but in particular. Of the inner surfaces of the container member 11 containing the culture solution containing cells, all the surfaces in contact with the cells (for example, the inner bottom surface and the inner surface of the container member 11) are either non-cell-adhesive or non-cell-adhesive. It is preferable that the sex can be changed.

The cell used in the present invention is not particularly limited as long as it is an adhesive cell. Examples of such cells include hepatocytes, cupper cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, fibroblasts, osteoblasts, crushed bone cells, and root membrane-derived cells, which are parenchymal cells of the liver. Epidermal cells such as epidermal keratinized cells, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells such as corneal epithelial cells, mammary gland cells, pericite, muscle cells such as smooth muscle cells and myocardial cells, renal cells , Pancreatic Langerhans islet cells, nerve cells such as peripheral nerve cells and optic nerve cells, cartilage cells, bone cells and the like. These cells may be primary cells taken directly from tissues or organs, or they may be passaged for several generations. Furthermore, these cells are undifferentiated embryonic stem cells, pluripotent mesenchymal stem cells and other pluripotent stem cells, monodifferentiable vascular endothelial precursor cells and other pluripotent stem cells, and differentiation has been completed. It may be any cell. In addition, the cells may be a single type of cells or two or more types of cells may be co-cultured.

These cells are cultured in advance by a usual method, the culture is treated by trypsin treatment or the like, and the cells are contained in the cell culture vessel 1 in a state of being suspended in a culture solution. As the culture medium, any cell culture medium 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., published by Asakura Shoten. A basal medium as described on page 581 of "Tissue Culture Technology Third Edition" edited by the Japan Society for Tissue Culture can be used. Further, serum (fetal bovine serum, etc.), various growth factors, antibiotics, amino acids, etc. may be added to the basal medium. In addition, 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 laminate, it is preferable to use a medium containing no 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-adhesion is determined by whether or not cell adhesion or elongation is unlikely to occur due to the chemical or physical properties of the surface. Specifically, the cell adhesion extension rate at the time of actual cell culture is used as an index, and 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 extension rate is determined by seeding ^{cells to be cultured within a seeding density range of 4000 cells / cm 2} or more and less than 30000 cells / cm ^{2 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 are adherently extended when they are stored in the cell and cultured for 14.5 hours ({(the number of adherent cells) / (the 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 the cells non-adhesive can be appropriately applied. In the case of cells with strong adhesive strength (for example, canine kidney tubule epithelial cells (MDCK)), if Petridish is used as it is, the cell laminate may adhere to the inner surface of the bottom, making it difficult to collect. , It is preferable to perform surface treatment to form a cell non-adhesive 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 whose cell adhesion has been improved by hydrophilic treatment such as plasma treatment or corona treatment is not preferable. The surface properties can be examined by measuring the O component of 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 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 but may be a double bond. Examples of the carbon-oxygen bond include a C—O bond, a C (= O) -O bond, a C = O bond, and the like.

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, surfactants, and amphipathic substances. These are physically or chemically crosslinked with each other and physically or chemically bonded to the bottom 10 which is a base material to form a hydrophilic film.

Specific examples of the water-soluble polymer material 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, and zwitterionic polymers. , Polysaccharides and the like. Examples of the molecular shape include linear, branched, and dendrimers. 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), copolymers of methacryloyloxyethylphosphorylcholine and 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, saponified products of vinyl acetate oligomers and their derivatives. Examples include derivatives, oligomers composed of diionic monomers and their derivatives, acrylic acid and its derivatives, methacrylic acid and its derivatives, acrylamide and its derivatives, biionic compounds, water-soluble silane coupling agents, water-soluble thiol compounds, and the like. Can be done. More specifically, ethylene glycol oligomer, (N-isopropylacrylamide) oligomer, methacryloyloxyethyl phosphorylcholine oligomer, low molecular weight dextran, low molecular weight heparin, oligoethylene glycol thiol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene. Glycol, 2- [methoxy (polyethyleneoxy) -propyl] trimethoxysilane, triethylene glycol-terminated-thiol and the like can be mentioned, but are not limited thereto.

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, and most preferably 1.5 nm to 1 μm. When the average thickness is 0.8 nm or more, it is not easily affected by the inner surface 101 of the bottom 10, which is preferable. Further, when 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 or the like) of the hydrophilic film on the inner surface 101 or directly coating the inner surface 101 with the hydrophilic organic compound or the like. Method, a method of coating the inner surface 101 with a hydrophilic organic compound or the like and then performing a cross-linking treatment, a method of forming a hydrophilic film in a multi-step manner in order to improve the adhesion to the inner surface 101, and improving the adhesion to the bottom 10. Therefore, 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 start point on the inner surface 101, and then a method of polymerizing a hydrophilic polymer brush can be mentioned.

Among the above-mentioned film forming methods, a particularly preferable method is a method of forming a hydrophilic film in a multi-step manner, and a method of forming an underlayer on the inner surface 101 in order to improve adhesion to the bottom 10, and then a hydrophilic organic substance. Examples thereof include a method of coating a compound or the like. This is because it is easy to improve the adhesion of the hydrophilic organic compound or the like to the bottom 10 by using these methods. The term "bonding layer" is used herein. The bonding layer means a layer existing between the outermost hydrophilic film layer and the bottom 10 when forming a film of a hydrophilic organic compound or the like in a multi-step manner, and an underlayer is provided on the inner surface 101. When a hydrophilic film layer is formed on the base layer, it means the base layer. The binding layer is preferably a layer containing a material having a binding moiety (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 an epoxy group and a hydroxyl group, a phthalic acid anhydride and a hydroxyl group, a carboxyl group and an N-hydroxysucciimide, a carboxyl group and a carbodiimide, an amino group and a glutaraldehyde, etc. Can be mentioned. In each combination, either may be a linker. In these methods, a binding layer is formed with a material having a linker on the inner surface 101 before coating with a hydrophilic material. The density of the material in the bond layer is an important factor that determines the bond strength. The density can be easily evaluated by using the contact angle of water on the surface of the bonding layer as an index. For example, in the case of a silane coupling agent (epoxysilane) having an epoxy group at the end, if the water contact angle of the inner surface 101 to which epoxysilane is added is typically 45 ° or more, preferably 47 ° or more. Next, by adding an ethylene glycol-based material or the like in the presence of an acid catalyst, a surface having sufficient cell non-adhesiveness can be obtained.

The inner surface 101 of the bottom 10 of the cell culture vessel 1 used in the present invention is preferably non-adhesive from the viewpoint of easy peeling of the cell laminate, but as another form, it is cell-adhesive during cell culture. However, it may be a surface capable of changing to cell non-adhesiveness upon exfoliation. Such a surface is for immobilizing at least one stimulus-responsive polymer selected from the group consisting of temperature-responsive polymers, pH-responsive polymers and ion-responsive polymers on the inner surface 101 via covalent bonds. Can be formed by The stimulus-responsive polymer is particularly preferably, but not limited to, a temperature-responsive polymer.

The temperature-responsive polymer that can be suitably used in the present invention exhibits hydrophobicity under the cell culture temperature (usually about 37 ° C.) and hydrophilicity under 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 (critical dissolution temperature (T) in water) is not particularly limited, but from the viewpoint of ease of recovery of the cell laminate, the cell culture temperature. It is preferable that the temperature is lower than that. By including such a temperature-responsive polymer component, sufficient cell scaffolding (cell adhesion surface) is secured at the time of cell culture, so that cell culture can be efficiently performed. On the other hand, at the time of collecting the cell laminate, the hydrophobic portion is changed to hydrophilic and the cell laminate is separated from the bottom 10, so that the recovery of the cell laminate can be further facilitated. In particular, a temperature-responsive polymer that exhibits hydrophilicity at a temperature lower than a predetermined critical dissolution temperature and hydrophobicity at a temperature equal to or higher than the predetermined temperature is preferable. The critical melting temperature of such a temperature-responsive polymer is particularly called the lower limit critical melting temperature.

Specifically, the temperature-responsive polymer that can be suitably used in the present invention is preferably a polymer having a lower limit critical dissolution temperature T of 0 ° C. to 80 ° C., preferably 0 ° C. to 50 ° C. If T exceeds 80 ° C., cells may die, which is not preferable. Further, when T is lower than 0 ° C., the cell growth rate is generally extremely lowered or the cells die, which is not preferable. Such suitable polymers include acrylic polymers 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-ethoxyethylacrylamide (T = about 35 ° C.), Poly-N-tetrahydrofurfurylacrylamide (T = about 28 ° C.), Poly-N-tetrahydrofurfurylmethacrylamide (T = about 35 ° C.), and Examples thereof include poly-N and N-diethylacrylamide (T = 32 ° C.). Other polymers include, for example, poly-N-ethylacrylamide, poly-N-isopropylmethacrylamide, poly-N-cyclopropylacrylamide, poly-N-cyclopropylmethacrylamide, poly-N-acryloylpyrrolidin, poly-N. -Alkyl substituted cellulose derivatives such as acryloyl piperidine, polymethyl vinyl ether, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, polyalkylene oxide block copolymers typified by block copolymers of polypropylene oxide and polyethylene oxide, and poly. Examples include alkylene oxide block copolymers.

Examples of the monomer for forming these polymers include a monomer in which the homopolymer of the monomer has T = 0 ° C. to 80 ° C. and can be polymerized by irradiation. Examples of the monomer include a (meth) acrylamide compound, an N- (or N, N-di) alkyl-substituted (meth) acrylamide derivative, a (meth) acrylamide derivative having a cyclic group, a vinyl ether derivative, and the like. 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 included in the present invention. When it is necessary to regulate T depending on the type of proliferating cells, copolymerization may be carried out by further adding monomers other than the above. 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. It is also possible to crosslink the polymer as long as 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. 2. Cell culture step The cell culture step is a cell culture under the condition that cell-cell adhesion is formed while applying a centrifugal force toward the inner surface 101 of the bottom 10 to the cells in the culture solution contained in the cell culture container 1. This is a step of adhering cells to each other to form a cell laminate. By this step, as shown in FIG. 2, the cell laminate 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 a cell laminate can be formed without adversely affecting the function of the cells. For example, the range of 2G to 1440G is preferable, and the range of 2G to 720G is more preferable. Centrifugal force can be applied by placing the cell culture vessel 1 containing the cell-containing culture solution in a centrifuge and performing a centrifugal operation.

The "condition under which cell-cell adhesion is formed" refers to a condition in which cells are active 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 to 24 hours is preferred. By applying 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 an extracellular matrix, and the culturing time can be 0.5 hours to 3 hours. When the extracellular matrix is not added, the culture time can be 1 hour to 24 hours.

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

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

3. 3. Placement step In the placement step, after the centrifugation operation is completed, as shown in FIG. 3, the positions of the bottom portion 10 and the top portion 20 are reversed, and the obtained cell laminate A is peeled off from the inner surface 101 of the bottom portion 10 and the top portion 20 is formed. It is a process of placing on. If the inner surface 101 is a non-adhesive surface of cells, the peeling operation is easy, and if necessary, an instrument such as tweezers is used as an auxiliary, or the cell laminate A is moved to the upper 20 side by the action of gravity. Can be made to. Further, when the inner surface 101 is a surface such as a stimulus-responsive polymer that can be changed to cell non-adhesive, an environment that becomes cell non-adhesive (for example, a temperature below the lower limit critical temperature). ) Is changed to the peeling operation.

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 laminate 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 laminate A is placed on the upper portion 20, and in this state, the cell laminate A can be easily transferred to, for example, a site where cell transplantation or the like is performed. At that time, since the cell laminate A is handled via the upper portion 20, it is possible to prevent the cell laminate A from being destroyed 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), make a circular through hole in the lid of this Petri dish with an ultrasonic cutter to an appropriate size (diameter about 25 to 30 mm), and then cover the lid. The edges were impregnated with toluene so that the film could adhere. Next, a biaxially stretched polystyrene film (OPS film; manufactured by Asahi Kasei Chemicals Co., Ltd.) was cut into a circle having a size of 35 mm and adhered to the lid to cover the through hole to prepare a cell culture vessel of Example 1. This cell culture vessel was sterilized by 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 at 37 ° C. for 10 minutes. Was gently peeled off from the substrate. ^{The proliferated cells were seeded in 1.5 × 10 7} cells in the above cell culture vessel and centrifuged at a centrifugal force of 50 G for 12 hours to form a cell laminate on the bottom surface of the Petri dish. Subsequently, when the cell culture vessel 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 impregnating the edge of the lid with toluene and adhering the OPS film, it was carried out except that the OPS film was adhered using a biocompatible deacetic acid type adhesive RTV118 (manufactured by Momentive Performance Materials). A cell culture vessel was prepared in the same manner as in Example 1. Adhesion was performed by leaving it in a room temperature environment for 2 days or more.

A cell laminate was prepared using this cell culture vessel, and the cell laminate could be transferred in a state of being placed on an 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 hollowed out in a circular shape by an ultrasonic cutter, and the same OPS film as in Example 1 was adhered to the portion by a solvent welding method using toluene. To prepare a cell culture vessel. Further, as Comparative Example 2, a cell culture vessel was prepared in the same manner as in Comparative Example 1 except that the OPS film was adhered using the same adhesive as in Example 2.

When an attempt was made to prepare a cell laminate by centrifugation using the cell culture vessels of Comparative Examples 1 and 2, the culture solution leaked from the gap between the bottom surface of the Petri dish and the OPS film after centrifugation, 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 prepare a cell culture container of Comparative Example 3. Using this cell culture vessel, an attempt was made to prepare a cell laminate by centrifugation.

As a result, when the size of the film to be laid underneath was smaller than the bottom surface of the Petri dish, cells stayed between the film and the bottom surface of the dish, and a cell laminate could not be produced. Further, 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 has not been specially processed is used, a cell laminate is formed by centrifugation on the bottom surface of the dish, and then the cell laminate is scooped with a film to transfer the cell laminate. I examined whether it could be done.

As a result, although the cell laminate was formed, it was difficult and time-consuming to capture the floating cell laminate when scooping with the film. In addition, a phenomenon of twisting when scooping the cell laminate or a phenomenon of destroying the cell laminate was observed.

(Weight measurement of cell laminate)
The following experiments were carried out to confirm that the present invention is useful in forming an appropriate tissue of a cell laminate.

First, a cell culture vessel was prepared by the procedure of Example 1 and Comparative Example 1 above using an OPS film whose weight was measured in advance, and a centrifugal force was applied under the same conditions as in Example 1 to form a cell laminate. Subsequently, in the cell culture vessel according to Example 1, the cell culture vessel was inverted after centrifugation, the culture solution was sucked out with a pipette, the OPS film was separated from the Petri dish lid, and the cell laminate was separated from the OPS film. Collected while placed on top. In the cell culture vessel 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 laminate 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 from the weight of the OPS film measured in advance was calculated to obtain the weight of the cell laminate alone.

As a result, when the cell culture vessel of Example 1 was used, a sufficient amount of cell laminate 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 surface of the Petri dish and the OPS film due to 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 clarified that the cell culture vessel of the present invention is useful for forming a sufficient amount of cell tissue as a cell laminate.

(Changes in cell viability depending on film type)
The following experiments were conducted to examine whether the viability of the cultured cells differed depending on the type of resin film adhered to the Petri dish lid.

As resin films covering the through holes formed in the lid of the petridish, PET film (manufactured by Asahi Kasei Chemicals Co., Ltd .; oxygen permeability 14.1 cc / m ² day atm) and OPS film (manufactured by Asahi Kasei Chemicals Co., Ltd .; oxygen permeation), respectively. Degree 50.3 cc / m ² · day · atm), polypropylene film (pyrene film-OT; manufactured by Toyo Boseki Co., Ltd .; 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 (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. In addition, as a comparison target, a cell laminate was formed by centrifugation using a normal Petri dish. 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 transmittance measuring device (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 laminate with PBS, 1 μg / ml Calcein-AM (Dojin Chemical Co., Ltd.) that stains live cells, 1 μg / ml Propidium Iodide (PI; Dojin Chemical Co., Ltd.) that stains dead cells, and Hoechst 33342 (diluted 1/1000; Invitrogen), which 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 with a confocal microscope.

The results are shown in Table 1. In Table 1, ○ means that the number of cells is about the same as that of the control (“dish”), Δ is slightly inferior, and × means that there are many dead cells. From this result, it was found that there is a difference in the cell viability after centrifugation depending on the type of resin film. The polyethylene film having high oxygen permeability was unsuitable 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.

(Effect of cell type)
The following experiments were conducted to investigate whether the state of the obtained cell laminate changes depending on the cell type.

Using CCL163 cells of mouse fibroblasts and MK442 (Takara Bio Inc.) of rat chondrocytes, a cell laminate was prepared and recovered from the cell culture vessel of Example 1. Incidentally, seeding cell numbers both with 1.5 × 10 ⁷ cells, as the resin film that adheres to the petri dish lid adopts PET film was bonded by an adhesive.

As a result of the experiment, a cell laminate was similarly obtained for the above two types of cells, and the handleability was excellent, and the cell laminate could be transferred without being destroyed or deformed.

Next, a similar study was conducted using MDCK cells with strong adhesive strength. Three cell culture containers are used as container members: Petri dish (manufactured by BD), polyethylene glycol (PEG) coated dish, and 3.5 cm dish of HydroCell (manufactured by CellSeed), and through holes are formed in these lids. , The through hole was covered with a PET film. The PEG-coated dish was manufactured by coating the surface with PEG400 (manufactured by Sanyo Chemical Industries, Ltd.) in accordance with the method described in Japanese Patent No. 5252036. HydroCell is a dish that has been surface-treated to make it non-adhesive to cells by surface-coating it with an acrylamide-based substance.

First, the cells grown in the medium were reacted with a 0.25% trypsin solution at 37 ° C. for 10 minutes. Cell seeding rate for cell culture vessel was 10 ⁷ 1.5 ×, the cell stack formed by centrifugation for 12 hours at 50G, cells laminated while placing the PET film by inverting the cell culture vessel We examined whether the body would be recovered. The results are shown in Table 2. In Table 2, ◯ indicates that the cell laminate was properly recovered, and × indicates that the recovery was difficult.

As shown in Table 2, the bottom surface of the Petri dish had an insufficient degree of cell non-adhesion to MDCK cells, and the cell laminate adhered to the bottom surface of the Petri dish, making it difficult to recover to the PET film. .. Naturally, the handleability of the cell laminate was also poor. On the other hand, in the two dishes whose surface was coated with the hydrophilic polymer, the formed cell laminate was suspended after centrifugation, and it was easy to recover to the PET film.

1 Cell culture container 10 Bottom 101 Inner surface 11 Container member 20 Top 21 Lid member 210 Through hole A Cell laminate

Claims (4)

A cell culture vessel having a bottom having an inner surface that is non-cell-adhesive or can be transformed into non-cell-adhesive, an upper portion facing the bottom, and a lid member that separably supports the upper portion. In the cell addition step of putting the culture solution containing cells into
A cell culture step in which cells are cultured under conditions where cell-cell adhesion is formed while applying centrifugal force toward the inner surface of the bottom of the cells, and the cells are adhered to form a cell laminate.
A mounting step of reversing the positions of the bottom and the top, peeling the obtained cell laminate from the inner surface, and placing the cell laminate on the top.
A separation step of separating the upper part from the lid member together with the placed cell laminate,
A method for producing a cell laminate having. The method for producing a cell laminate according to claim 1, wherein the upper part is a film. The method for producing a cell laminate according to claim 2, wherein the film is a polyethylene terephthalate film or a laminated film containing the polyethylene terephthalate film. The method for producing a cell laminate according to claim 1, wherein the upper part has an oxygen permeability of 200 cc / m ^{2, day, atm or less.}