JP2018201394A - Methods for preparing cell sheet - Google Patents

Abstract

To provide methods for preparing cell sheet that enables easy peeling of the formed cell sheet adhering to the culture vessel with minimum damage to the cell sheet.SOLUTION: Disclosed herein is a method for producing a cell sheet comprising: growing cells adhered to a culture surface with a liquid culture medium in a culture vessel until a cell sheet is formed; and peeling the grown cell sheet from the culture vessel using a peeling solution containing at least a chelating agent but no protease, where the culture vessel, at least the portion to which the cell sheet adheres, is formed with a polymer having an alycyclic structure.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing a cell sheet, and more particularly, to a method for producing a cell sheet formed of cells that grow by adhering to a culture vessel.

  In recent years, cell sheets used for regenerative medicine and the like have been developed. In order to peel off the cell sheet formed on the bottom surface of the culture container so that the cells are not damaged as much as possible, it has been studied to form a temperature sensitive substrate or a light sensitive substrate on the bottom surface of the culture container (Patent Document 1). ~ 4).

  However, although any of these methods can reduce the damage to cells, it is inferior in productivity, such as requiring a new culture step. In addition, a method of physically peeling the cells from the bottom of the culture vessel using a scraper or the like is also performed, but there is a concern that the stress received by the cells is too great.

  By the way, Patent Document 5 reports that in cell culture using a cycloolefin resin container, cell proliferation is improved as compared with the case where a polystyrene container is used. In the Examples, it is shown that the proliferation of blood cells that are not adherent cells is improved in an environment coated with a protein such as anti-CD3 antibody or retronectin.

JP 2017-025335 A JP 2006-013111 A JP, 2006-079268, A JP 2012-044905 A JP 2008-048553 A

  The present invention has been made in view of the above-described circumstances, and is a cell sheet capable of easily detaching a cell sheet formed by adhering to a culture container while suppressing damage to the cell as much as possible. It aims at providing the manufacturing method of.

  The inventor has intensively studied to achieve the above object. As a result, when a culture vessel made of an alicyclic structure-containing polymer is used as the culture vessel, the cells that have grown by adhering to the culture vessel can be easily peeled off and collected just by using a chelating agent as the cell peeling agent. The present inventors have found that this can be done and have completed the present invention.

That is, the present invention aims to advantageously solve the above-mentioned problems, and the method for producing a cell sheet according to the present invention adheres to a culture surface of the culture container in a culture container containing a liquid medium. The cell sheet is proliferated until it becomes a sheet on the culture surface, and the proliferated sheet-like cell is detached from the culture surface with a peeling solution that does not contain a proteolytic enzyme and contains at least a chelating agent. In the production method, at least the culture surface of the culture vessel is formed of an alicyclic structure-containing polymer.
If the culture surface is a culture vessel formed of an alicyclic structure-containing polymer, cells attached to the culture surface can be easily detached in the form of a sheet simply by using a stripping solution containing a chelating agent. . Therefore, the cell sheet can be peeled and collected without reducing the productivity. In addition, since no tools such as scrapers or proteolytic enzymes are used, damage to cells can be reduced as much as possible.

  In the method for producing a cell sheet of the present invention, the chelating agent is preferably ethylenediaminetetraacetic acid. This is because, among the chelating agents, when ethylenediaminetetraacetic acid (EDTA) is used as the release agent, the release property of the cell sheet is particularly good.

  Furthermore, in the method for producing a cell sheet of the present invention, the water contact angle on the culture surface of the culture vessel is preferably 85 ° or more and 110 ° or less. If the water contact angle is 85 ° or more and 110 ° or less, the peelability of the cell sheet is particularly good.

  According to the method for producing a cell sheet of the present invention, a cell sheet formed by adhering to a culture container can be easily detached from the culture container while minimizing damage to the cells.

It is a photograph which shows a mode that the cell has peeled from the outer periphery in the sheet form. It is a photograph which shows a mode that the cell has peeled from the outside in the sheet form. It is a photograph which shows a mode that the cell has peeled from the outside in the sheet form. It is a photograph which shows a mode that the cell has peeled from the outside in the sheet form. It is the photograph which carried out Oil Red dyeing | staining of the cell sheet. It is the photograph which stained the cell sheet with Alizarin Red. It is the photograph which stained the cell sheet for Alcian Blue. It is a photograph which shows a mode that the cell has peeled from the outside in the sheet form. It is the photograph of the cell sheet which peeled from the culture container and moved to another culture container.

Hereinafter, embodiments of the present invention will be described in detail.
(Cell culture method)
In this embodiment, the culture medium in which the cells are suspended is transferred into the culture container, the culture container is left still, and the cells are adhered to the culture container and cultured. Examples of the portion where cells are adhered to form a sheet include the bottom surface of the culture vessel, but may be any surface or part as long as the cells can adhere and proliferate, and are not limited thereto.
<Cell>
The cells may be any cells as long as they grow by adhering to the culture vessel, and can be arbitrarily selected according to the purpose. Preferred cells include adipose stem cells (ASC), bone marrow stem cells (MSC), mesenchymal stem cells, hematopoietic stem cells, neural stem cells, skin stem cells, and other tissue stem cells (also referred to as adult stem cells and somatic stem cells); chondrocytes; osteoblasts Cells; hair papilla cells; and the like.

<Liquid medium>
The cells are cultured using a liquid medium.
As the liquid medium, a medium having a pH buffering action, having an osmotic pressure suitable for cells, containing nutrient components of cells, and not toxic to cells is used.
Examples of the component that imparts a pH buffering action to the liquid medium include Tris hydrochloride, various phosphates, and various carbonates.
The osmotic pressure of the liquid medium is usually adjusted using an aqueous solution in which the concentrations of potassium ions, sodium ions, calcium ions, glucose and the like are adjusted so as to be almost the same as the osmotic pressure of the cells. Specific examples of the aqueous solution include physiological saline such as phosphate buffered saline, Tris buffered saline, and HEPES buffered saline; Ringer's solution such as lactated Ringer's solution, acetated Ringer's solution, and bicarbonated Ringer's solution; It is done.
Examples of the nutrient component of the cell include amino acids, nucleic acids, vitamins, minerals and the like.
As the liquid medium, a commercially available medium suitable for various cells can be used.

<Additives>
An additive can also be mix | blended with a liquid culture medium.
Additives used include peptides; minerals; metals; vitamin components; ligands, agonists and antagonists that act on cell surface receptors; nuclear receptors, ligands, agonists and antagonists; extracellular such as collagen and fivenectin Matrix: A part of the extracellular matrix or a compound that mimics the extracellular matrix; a component that acts on a protein involved in a signal transduction pathway in a cell; a component that acts on an enzyme of primary or secondary metabolism in a cell; Components that affect gene expression in the nucleus or mitochondria.
These additives can be used singly or in combination of two or more, and are added according to the purpose of promoting cell proliferation or inducing differentiation into a predetermined cell.

<Cell culture conditions>
The cell culture conditions are not particularly limited, and can be appropriately determined according to the cells to be used and the purpose. For example, the cells can be cultured using a humidified thermostat having a carbon dioxide concentration of about 5% and a constant temperature in the range of 20 ° C to 37 ° C.

(Culture container)
In this embodiment, the culture vessel is formed by molding an alicyclic structure-containing polymer into an arbitrary shape. In the present invention, it is sufficient that at least the culture surface of the culture vessel is formed of an alicyclic structure-containing polymer.
The alicyclic structure-containing polymer is a resin having an alicyclic structure in the main chain and / or side chain, and preferably contains an alicyclic structure in the main chain from the viewpoint of mechanical strength, heat resistance, and the like.

<Alicyclic structure>
Examples of the alicyclic structure include a saturated cyclic hydrocarbon (cycloalkane) structure and an unsaturated cyclic hydrocarbon (cycloalkene) structure. From the viewpoint of mechanical strength, heat resistance, etc., a cycloalkane structure or a cycloalkene structure. Among them, those having a cycloalkane structure are most preferable.

  The number of carbon atoms constituting the alicyclic structure is not particularly limited, but is usually 4 to 30, preferably 5 to 20, and more preferably 5 to 15. When the number of carbon atoms constituting the alicyclic structure is within this range, the mechanical strength, heat resistance, and moldability characteristics are highly balanced, which is preferable.

  The proportion of the repeating unit having an alicyclic structure in the alicyclic structure-containing polymer may be appropriately selected according to the purpose of use, but is usually 30% by weight or more, preferably 50% by weight or more, more preferably 70% by weight. % Or more. If the proportion of the repeating unit having an alicyclic structure in the alicyclic structure-containing polymer is excessively small, the heat resistance is inferior, which is not preferable. The remainder other than the repeating unit having an alicyclic structure in the alicyclic structure-containing polymer is not particularly limited and is appropriately selected according to the purpose of use.

<Aricyclic structure-containing polymer>
Specific examples of the alicyclic structure-containing polymer include (1) a norbornene polymer, (2) a monocyclic olefin polymer, (3) a cyclic conjugated diene polymer, and (4) a vinyl alicyclic ring. And hydrocarbon hydrocarbon polymers. Among these, norbornene-based polymers are preferable from the viewpoints of heat resistance, mechanical strength, and the like.

(1) Norbornene-based polymer The norbornene-based polymer is obtained by polymerizing a norbornene-based monomer that is a monomer having a norbornene skeleton, and is obtained by ring-opening polymerization or by addition polymerization. Broadly divided into things.

Examples of the ring-opening polymer obtained by ring-opening polymerization include ring-opening polymers of norbornene monomers, ring-opening polymers of norbornene monomers and other monomers capable of ring-opening copolymerization, and these A hydride etc. are mentioned.
Examples of those obtained by addition polymerization include addition polymers of norbornene monomers and addition polymers of norbornene monomers and other monomers copolymerizable therewith.
Among these, ring-opening polymer hydrides of norbornene monomers, addition polymers of norbornene monomers and other monomers copolymerizable therewith, hydrogenated products of the addition polymers, etc. The saturated norbornene-based polymer is preferable from the viewpoints of heat resistance, mechanical strength, and the like, and from the viewpoint of ease of cell detachment, those having no functional group are particularly preferable. Here, the functional group means an atom or an atomic group having atoms other than carbon and hydrogen. Examples of functional groups include amino groups, carboxyl groups, hydroxyl groups, acid anhydride groups, and halogen atoms.

As the norbornene-based monomer, bicyclo [2.2.1] hept-2-ene (common name: norbornene), 5-methyl-bicyclo [2.2.1] hept-2-ene, 5,5- Dimethyl-bicyclo [2.2.1] hept-2-ene, 5-ethyl-bicyclo [2.2.1] hept-2-ene, 5-ethylidene-bicyclo [2.2.1] hept-2- Ene, 5-vinyl-bicyclo [2.2.1] hept-2-ene, 5-propenylbicyclo [2.2.1] hept-2-ene, 5-methoxycarbonyl-bicyclo [2.2.1] Bicyclic such as hepta-2-ene, 5-cyanobicyclo [2.2.1] hept-2-ene, 5-methyl-5-methoxycarbonyl-bicyclo [2.2.1] hept-2-ene Monomer; tricyclo [4.3.0 ^1,6 . 1 ^2,5 ] deca-3,7-diene (common name: dicyclopentadiene), 2-methyldicyclopentadiene, 2,3-dimethyldicyclopentadiene, 2,3-dihydroxydicyclopentadiene, etc. Monomer; Tetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene (tetracyclododecene), tetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-methyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-ethyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-ethylidenetetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8,9-dimethyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-ethyl-9-methyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-ethylidene-9-methyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-methyl-8-carboxymethyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 7,8-benzotricyclo [4.3.0.1 ^2,5 ] dec-3-ene (common name: methanotetrahydrofluorene: 1,4-methano-1, 4,4a, 9a-tetrahydrofluorene), 1,4-methano-8-methyl-1,4,4a, 9a-tetrahydrofluorene, 1,4-methano-8-chloro-1,4,4a, 9a -Tetracyclic monomers such as tetrahydrofluorene and 1,4-methano-8-bromo-1,4,4a, 9a-tetrahydrofluorene;

Other monomers capable of ring-opening copolymerization with norbornene monomers include cyclohexene, cycloheptene, cyclooctene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, 1,5-cyclodecadiene, And monocyclic cycloolefin monomers such as 1,5,9-cyclododecatriene and 1,5,9,13-cyclohexadecatetraene.
These monomers may have one or more substituents. Examples of the substituent include an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, and an alkylidene group.

Other monomers capable of addition copolymerization with a norbornene monomer include α-olefin monomers having 2 to 20 carbon atoms such as ethylene, propylene, 1-butene, 1-pentene and 1-hexene; cyclobutene, cyclopentene, cyclohexene, cyclooctene, tetracyclo ^{[9.2.1.0 2,10.} Cycloolefin monomers such as 0 ^3,8 ] tetradeca-3,5,7,12-tetraene (also referred to as 3a, 5,6,7a-tetrahydro-4,7-methano-1H-indene); And non-conjugated diene monomers such as 4-hexadiene, 4-methyl-1,4-hexadiene, 5-methyl-1,4-hexadiene, and 1,7-octadiene.
Among these, as other monomers that can be addition copolymerized with norbornene monomers, α-olefin monomers are preferable, and ethylene is more preferable.
These monomers may have one or more substituents. Examples of the substituent include an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, and an alkylidene group.

A ring-opening polymer of a norbornene-based monomer, or a ring-opening polymer of a norbornene-based monomer and another monomer capable of ring-opening copolymerization with a monomer component is a known ring-opening polymerization. It can be obtained by polymerization in the presence of a catalyst. Examples of the ring-opening polymerization catalyst include a catalyst comprising a metal halide such as ruthenium or osmium, a nitrate or an acetylacetone compound, and a reducing agent, or a metal halide or acetylacetone such as titanium, zirconium, tungsten or molybdenum. A catalyst comprising a compound and an organoaluminum compound can be used.
The ring-opening polymer hydride of a norbornene-based monomer is usually obtained by adding a known hydrogenation catalyst containing a transition metal such as nickel or palladium to the polymerization solution of the above-described ring-opening polymer, and then adding a carbon-carbon unsaturated bond. Can be obtained by hydrogenation.

  An addition polymer of a norbornene-based monomer or an addition polymer of a norbornene-based monomer and another monomer copolymerizable therewith is a monomer component in the presence of a known addition polymerization catalyst. It can be obtained by polymerization. As the addition polymerization catalyst, for example, a catalyst composed of a titanium, zirconium or vanadium compound and an organoaluminum compound can be used.

(2) Monocyclic Cyclic Olefin Polymer Examples of the monocyclic cycloolefin polymer include addition polymers of monocyclic cycloolefin monomers such as cyclohexene, cycloheptene, and cyclooctene. .
(3) Cyclic conjugated diene polymer As the cyclic conjugated diene polymer, for example, a polymer obtained by subjecting a cyclic conjugated diene monomer such as cyclopentadiene or cyclohexadiene to 1,2- or 1,4-addition polymerization, and The hydride etc. are mentioned.
(4) Vinyl alicyclic hydrocarbon polymer Examples of the vinyl alicyclic hydrocarbon polymer include polymers of vinyl alicyclic hydrocarbon monomers such as vinylcyclohexene and vinylcyclohexane, and hydrogenated products thereof; styrene; , Hydrides of aromatic ring portions of polymers of vinyl aromatic monomers such as α-methylstyrene; and the like.
The vinyl alicyclic hydrocarbon polymer may be a copolymer with other monomers copolymerizable with these monomers.
These alicyclic structure-containing polymers can be used alone or in combination of two or more.

  Although there is no special restriction | limiting in the molecular weight of an alicyclic structure containing polymer, it is a weight average molecular weight of polystyrene conversion measured by the gel permeation chromatography of a cyclohexane solution (a toluene solution when a polymer does not melt | dissolve), and is usually 5 5,000 or more, preferably 5,000 to 500,000, more preferably 8,000 to 200,000, and particularly preferably 10,000 to 100,000. When the weight average molecular weight is within this range, the mechanical strength and the moldability are highly balanced, which is preferable.

  The glass transition temperature of the alicyclic structure-containing polymer may be appropriately selected depending on the purpose of use, but is usually 50 to 300 ° C, preferably 100 to 280 ° C, particularly preferably 115 to 250 ° C, and more preferably 130. ~ 200 ° C. When the glass transition temperature is within this range, heat resistance and molding processability are highly balanced and suitable. In addition, the glass transition temperature of the said alicyclic structure containing polymer is measured based on JISK7121.

For the alicyclic structure-containing polymer, compounding agents usually used in thermoplastic resin materials, for example, soft polymers, antioxidants, ultraviolet absorbers, light stabilizers, near infrared absorbers, mold release agents, dyes Coloring agents such as pigments and pigments, plasticizers, antistatic agents, fluorescent whitening agents, and other compounding agents can be added in the amounts usually employed.
In addition, the alicyclic structure-containing polymer may be mixed with another polymer other than the soft polymer (hereinafter simply referred to as “other polymer”). The amount of the other polymer mixed with the alicyclic structure-containing polymer is usually 200 parts by weight or less, preferably 150 parts by weight or less, more preferably 100 parts by weight with respect to 100 parts by weight of the alicyclic structure-containing polymer. It is as follows.
If the proportion of various compounding agents and other polymers to be blended with the alicyclic structure-containing polymer is too high, cells will not float easily, so all are blended within the range that does not impair the properties of the alicyclic structure-containing polymer. It is preferable.

  The mixing method of the alicyclic structure-containing polymer and the compounding agent or other polymer is not particularly limited as long as the compounding agent is sufficiently dispersed in the polymer. Moreover, there is no special restriction | limiting in a mixing | blending order. As a mixing method, for example, a method of kneading a resin in a molten state using a mixer, a single-screw kneader, a twin-screw kneader, a roll, a Brabender, an extruder, etc., after dissolving and dispersing in an appropriate solvent, Examples thereof include a method of removing the solvent by a coagulation method, a casting method, or a direct drying method. When a biaxial kneader is used, after kneading, it is usually extruded in a rod shape in a molten state, cut into an appropriate length with a strand cutter, and pelletized in many cases.

<Molding of culture container>
The method for forming the culture vessel can be arbitrarily selected according to the shape of the culture vessel. Specific examples of molding methods include injection molding, extrusion molding, cast molding, inflation molding, blow molding, vacuum molding, press molding, compression molding, rotational molding, calendar molding, rolling molding. Method, cutting molding method, spinning, and the like. These molding methods can be combined, or post-treatment such as stretching can be performed as necessary after molding.

Further, the culture vessel may be any surface as long as the cell adheres, that is, the culture surface is composed of an alicyclic structure-containing polymer molded body, and the entire culture vessel is formed of the alicyclic structure-containing polymer. It doesn't have to be a thing.
That is, the culture container may be a container containing the alicyclic structure-containing polymer molded body as a part of the constituent member, or may be a container composed entirely of the alicyclic structure-containing polymer molded body. And the container comprised by the laminated body of an alicyclic structure containing polymer molded object and another polymer molded object may be sufficient.

There is no particular limitation on the shape of the polymer molded product containing an alicyclic structure that forms a culture surface on which cells adhere and grow, and examples thereof include a plate shape and a sheet shape, and even if the surface is flat, You may have uneven | corrugated shape.
Examples of the shape of the culture container include a dish, a plate, a bag, a tube, a scaffold, a cup, and a jar fermenter.

<Sterilization of culture container>
The culture container is preferably sterilized before being used for cell culture.
There are no particular restrictions on the sterilization method, heating methods such as the high-pressure steam method and dry heat method, radiation methods that irradiate radiation such as γ rays and electron beams, irradiation methods that irradiate high frequencies, and ethylene oxide gas (EOG) The method can be selected according to the shape of the culture vessel and the cells to be used from methods generally employed in the medical field, such as a gas method in which a gas is brought into contact, a filtration method using a sterilization filter, and the like. Among these, the gas method is preferable because the change in the surface state is small.

<Surface treatment of culture surface>
In addition, the surface of the culture surface where cells adhere and grow is generally used for the purpose of imparting hydrophilicity to the inner surface of the container in order to promote cell adhesion, such as plasma treatment, corona discharge treatment, ozone treatment, and ultraviolet irradiation treatment. It is also possible to perform a hydrophilic treatment. However, the cost generated by performing these surface treatment operations can be suppressed; the cleanliness may be impaired by partial decomposition of the surface of the formed body accompanying the surface treatment; the peelability of the cell sheet may be reduced. For example, it is preferable that the hydrophilization treatment is not substantially performed because of the fact that the water contact angle range described later cannot be maintained.

<Water contact angle of culture surface>
The water contact angle of the culture surface on which the cells adhere and grow is preferably 85 ° to 110 °, more preferably 85 ° to 105 °, and more preferably 85 ° to 100 °. Particularly preferred. Here, the water contact angle is measured using a known fully automatic contact angle meter (for example, “LCD-400S” manufactured by Kyowa Interface Science Co., Ltd.), and the surface to be measured is cut with a circle cutter having a diameter of 30 mm, The radius r and height h of the droplet are obtained by measuring a total of five points of four vertices of a square having a side of 20 mm with the center as the center, and tan θ ₁ = h / r, θ = 2θ ₁ → θ = Θ calculated by 2 arctan (h / r) (θ / 2 method).

(Peeling of cell sheet)
In order to peel the cell sheet from the culture container, it is necessary to peel the cell sheet from the culture surface of the culture container. The cell sheet formed on the culture surface does not require a proteolytic enzyme and can be detached by adding a stripping solution containing at least a chelating agent. Among the chelating agents, when ethylenediaminetetraacetic acid (EDTA) is used as a release agent, the release property of the cell sheet is particularly good. In addition, when the culture surface of the culture vessel has the water contact angle described above, the peelability of the cells is further improved. To peel the cell sheet from the container, neither a proteolytic enzyme nor a device such as a scraper that directly applies the force to the cell is required.
<Cell sheet>
The cells are grown on almost the entire culture surface, that is, the culture is continued to 90% confluence or more, preferably 95% confluence or more, more preferably 100% confluence, so that the cells become a sheet. It is not preferable to culture the cells beyond 100% confluence because it promotes cell death.

<Release solution>
The stripping solution may contain a buffer solution such as physiological saline or PBS for the purpose of diluting the chelating agent in addition to the chelating agent, but does not contain a proteolytic enzyme such as trypsin. The concentration of the chelating agent contained in the stripper is preferably 0.1 to 10 mM. More preferably, it is 0.5-5 mM.
As the chelating agent contained in the stripping solution, aminocarboxylic acid chelating agents such as ethylenediaminetetraacetic acid (EDTA), glycol etherdiaminetetraacetic acid (EGTA), nitrilotriacetic acid (NTA), and diethylenetriaminepentaacetic acid (DTPA) are suitable. Take as an example. Among these, ethylenediaminetetraacetic acid (EDTA) is particularly preferable because of good peelability of the cell sheet.
The contact time between the peeling solution containing the chelating agent and the cell sheet is usually 1 to 20 minutes, preferably 1 to 15 minutes, more preferably 1 to 10 minutes.
There is no particular limitation on the temperature at the time of contact between the peeling solution containing the chelating agent and the cell sheet, but any temperature can be set in consideration of the cell survival rate and workability. Usually, it is 15-37 degreeC, Preferably it is 10-25 degreeC.
The cells that have come into contact with the stripping solution containing the chelating agent gradually peel off from the outer periphery, but can be uniformly detached by rubbing the cells with a pipettor tip along the outer periphery of the container. Scrape this with a jig such as a plastic film such as a circular polyvinylidene fluoride (PVDF) smaller than the diameter of the bottom of the petri dish or a wide spatula; be able to. The recovery method can be selected in consideration of the thickness, shape and other properties of the membrane.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples.
Example 1
Using a norbornene-based ring-opening polymer hydride (manufactured by Nippon Zeon Co., Ltd .; ZEONOR (registered trademark) 1060R) as the alicyclic structure-containing polymer, a petri dish-shaped culture vessel having an inner diameter of 10 cm was obtained by an injection molding method. Hereinafter, this culture container is referred to as “1060R dish”. Subsequently, the obtained culture container was sterilized with ethylene oxide. The water contact angle of the bottom surface (the side in contact with the cells) of the 1060R dish was 90 °.
Adipose-derived stem cells (ASC) suspended in 10 mL of Iscob modified Dulbecco medium (manufactured by Nacalai Tesque; hereinafter referred to as “IMDM”) are seeded in a 1060R dish so as to be 20% confluent, and 5% CO _{2. A} culture medium of adipose-derived stem cells (manufactured by Cosmo Bio Inc .; catalog number KBM ADSC-2) and IMDM, cultured at 37 ° C. in an atmosphere and added with 2% fetal bovine serum (FBS) after 12 hours Was replaced. Then cultured to 100% confluency in continued atmosphere of 5% CO ₂ 37 ° C. conditions. After removing the culture solution in the culture vessel by suction and washing with physiological saline, 5 mL of 1 mM ethylenediaminetetraacetic acid (EDTA) -containing physiological saline was added and allowed to stand for 10 to 20 minutes. It was confirmed that it had peeled off. FIG. 1 shows a photograph of the cells being peeled from the outer periphery in a sheet form. The peripheral part is a cell sheet that has been peeled off in white, and the central part is an area that is still adhered to the bottom of the container.

(Example 2)
The ASC medium cultured to 80% to 100% confluent state in the same manner as in Example 1 was prepared by using an adipocyte differentiation medium (DS Pharma Biomedical Co., Model No. BBDM2), an osteoblast differentiation medium (DS Pharma Biomedical Co., Ltd.). The model number BBOB1) and the chondrocyte differentiation medium (Thermo Fisher Scientific, StemPro Chronogenesis differentiation Kit; model number A10070-01) are replaced with a new medium every 3 to 4 days thereafter. Differentiation was induced by culturing for 2 weeks.
When various differentiation-induced cell sheets are added by the above-described method, that is, 5 mL of 1 mM EDTA-containing physiological saline and left to stand for 10 to 20 minutes, as shown in the photographs in FIGS. I confirmed that I started to do. In the photograph (64 ×) in FIG. 2, cells that have been induced to differentiate into adipocytes are starting to peel from the outside in the form of a sheet. In the photograph (64 ×) in FIG. 3, cells that have been induced to differentiate into osteoblasts are starting to peel from the outside in the form of sheets. In the photograph (64 ×) in FIG. 4, cells that have been induced to differentiate into chondrocytes are starting to peel from the outside in the form of a sheet.

Each of the cell sheets in the middle of detachment was fixed with 4% neutral formalin for 10 minutes, washed with distilled water, and the cell sheet obtained by culturing with adipocyte differentiation medium was subjected to Oil Red staining and osteoblast differentiation medium. The cell sheet obtained by the culture was stained by Alizarin Red staining, and the cell sheet obtained by the culture using the chondrocyte differentiation medium was stained by Alcian Blue staining. In the photograph of FIG. 5 (160 ×), cells that have been induced to differentiate into adipocytes form oil droplets, and the cell sheet is stained red with Oil Red. In the photograph of FIG. 6 (64 ×), a sheet of cells that have been induced to differentiate into osteoblasts is stained red with Alizarin Red. In the photograph (64 ×) in FIG. 7, the sheet of cells induced to differentiate into chondrocytes is stained blue with Alcian Blue.
From these results, it was found that differentiation was induced in the target cells corresponding to each differentiation-inducing medium, and a sheet composed of these differentiated cells was obtained.

Example 3
A 1060R internal diameter 6 cm dish was seeded with chondrocytes isolated from cartilage and cultured to 100% confluent as in Example 1 except that Dulbecco's modified Eagle (DMEM) medium supplemented with 10% fetal bovine serum (FBS) was used. And it processed similarly with 1 mM EDTA. As shown in the photograph (64 ×) in FIG. 8, chondrocytes began to peel into a sheet. As shown in the photograph of FIG. 9, the peeled cell sheet could be moved to the 1060R dish having the same inner diameter of 10 cm as used in Example 1.

(Comparative Example 1)
ASC was cultured in the same manner as in Example 1 except that it was a commercially available dish for adherent cells (manufactured by TruLine; model number TR4002; diameter 10 cm; made of polystyrene) instead of the 1060R dish. The culture solution in the culture vessel was removed by suction and washed with physiological saline, and then 5 mL of physiological saline containing 1 mM ethylenediaminetetraacetic acid (EDTA) was added and allowed to stand for 10 minutes, but the cell sheet was not peeled off.

  According to the method for producing a cell sheet of the present invention, there is provided a method for producing a cell sheet capable of easily detaching a cell sheet formed by adhering to a culture vessel while suppressing damage to the cell as much as possible. Can be provided.

Claims (3)

In a culture container containing a liquid medium, the cells adhered to the culture surface of the culture container are grown until they become a sheet on the culture surface,
A method for producing a cell sheet, in which the proliferated cells are exfoliated from the culture surface with a exfoliation solution that does not contain a proteolytic enzyme and contains at least a chelating agent,
A method for producing a cell sheet, wherein at least the culture surface of the culture vessel is formed of an alicyclic structure-containing polymer.   The method for producing a cell sheet according to claim 1, wherein the chelating agent is ethylenediaminetetraacetic acid.   The manufacturing method of the cell sheet of Claim 1 or 2 whose water contact angle of the culture surface of the said culture container is 85 degrees or more and 110 degrees or less.