JP2020018193A - Pluripotent stem cell detachment method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for exfoliating pluripotent stem cells, which is excellent in mass productivity, exfoliates pluripotent stem cells with a high survival rate, and can be recovered as a single cell. A method for exfoliating pluripotent stem cells cultured on a substrate, wherein the substrate has a layer made of a temperature-responsive polymer having a product of layer thickness x content ratio of temperature-responsive components of 100 nm or less. A method for exfoliating pluripotent stem cells, which comprises the following steps [1] to [3]. [1] A step of contacting a pluripotent stem cell cultured on a substrate with an intercellular binding dissociation solution containing trypsin. [2] A step of removing the intercellular binding divergent solution containing trypsin while the pluripotent stem cells are adhered to the substrate. [3] A step of cooling the substrate and exfoliating pluripotent stem cells from the substrate. [Selection diagram] None

Description

  The present invention relates to a method for exfoliating pluripotent stem cells, which excels in mass productivity, exfoliates pluripotent stem cells at a high survival rate, and can be recovered as a single cell.

  Pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) have the ability to differentiate into various tissues of the living body (pluripotency), and are cells in the field of regenerative medicine. As a source, great attention is being paid. In order to apply pluripotent stem cells to regenerative medicine, it is essential to first grow a required number of pluripotent stem cells in an undifferentiated state and then collect them as single cells.

  Conventionally, in order to collect pluripotent stem cells cultured on a substrate as single cells, the cells are treated with an enzyme such as trypsin and then rubbed with a cell scraper to peel the pluripotent stem cells from the substrate. I was letting it. However, in such a method for detaching pluripotent stem cells, it is difficult to collect a large amount of cells using a large-area substrate because the entire substrate must be rubbed with a cell scraper.

  As another method of exfoliating cells without using a cell scraper, a method using a substrate having temperature responsiveness is known. For example, in Patent Document 1, a cell culture support coated with a temperature-responsive polymer having an upper or lower critical dissolution temperature in water of 0 to 80 ° C. is used, cells are detached by a change in temperature, and a cell sheet is collected. A method for doing so is disclosed. However, the method described in Patent Literature 1 is mainly intended to recover a cell sheet, and cannot obtain a single cell pluripotent stem cell.

JP 2009-131275 A

  An object of the present invention is to provide a method for exfoliating pluripotent stem cells, which excels in mass productivity, exfoliates pluripotent stem cells at a high survival rate, and can be recovered as a single cell.

  In view of the above points, the present inventors have conducted intensive studies, and as a result, using a substrate having a layer of a temperature-responsive polymer having a product of layer thickness x content of the temperature-responsive component of 100 nm or less, trypsin It has been found that the above problem can be solved by a step of contacting a pluripotent stem cell with an intercellular binding dissociation solution containing the present invention, and the present invention has been completed.

That is, the present invention is a method for exfoliating pluripotent stem cells cultured on a substrate, wherein the substrate comprises a layer of a temperature-responsive polymer having a product of a layer thickness x a content ratio of a temperature-responsive component of 100 nm or less. A method for exfoliating pluripotent stem cells, comprising the steps [1] to [3] below.
[1] A step of contacting a pluripotent stem cell cultured on a substrate with an intercellular binding dissociation solution containing trypsin.
[2] A step of removing the trypsin-containing cell-binding dissociation solution while the pluripotent stem cells adhere to the substrate.
[3] A step of cooling the substrate and peeling the pluripotent stem cells from the substrate.

  According to the present invention, it is possible to provide a method for exfoliating pluripotent stem cells, which is excellent in mass productivity and can exfoliate pluripotent stem cells as a single cell.

  Hereinafter, embodiments for carrying out the present invention (hereinafter, simply referred to as “the present embodiment”) will be described in detail. The following embodiment is an exemplification for describing the present invention, and is not intended to limit the present invention to the following contents. The present invention can be appropriately modified and implemented within the spirit of the invention.

  As used herein, the term “temperature-responsive polymer” refers to a polymer whose degree of hydrophilicity / hydrophobicity changes with a change in temperature. In addition, when the temperature of a polymer is raised from low to high in the presence of water molecules, the degree of hydrophilicity / hydrophobicity of the polymer changes to more hydrophobic at a certain temperature, and the water solubility is increased. There is a temperature-responsive polymer that changes from water-insoluble to water-insoluble, and this boundary temperature is referred to as “Lower Critical Solution Temperature (LCST)”. In general, when a temperature-responsive polymer dissolves in water at a temperature lower than LCST, it becomes insoluble in water at a temperature higher than LCST. When the temperature-responsive polymer is water-insoluble, it has no LCST, but has a response temperature at which the degree of hydrophilicity / hydrophobicity changes with temperature change.

  In the present invention, the type of pluripotent stem cells is not particularly limited. For example, pulmonary stem cells (ES cells), induced pluripotent stem cells (iPS cells), and nuclear transplanted ES cells (ntES cells) are used. And particularly preferably iPS cells. The animal from which the pluripotent stem cells are derived is not particularly limited, but may be, for example, a mammal. Examples of mammals include rodents (mouse, rat, etc.) and primates (monkey, human, etc.), and may be experimental animals or companion animals. In the present invention, it is preferably derived from a primate, and particularly preferably derived from a human.

  In the step [1] of the method for detaching a pluripotent stem cell of the present invention, the pluripotent stem cell is brought into contact with a cell-binding dissociation solution. By bringing the pluripotent stem cell into contact with the cell-cell dissociation solution, the cell-cell junction of the pluripotent stem cell can be dissociated, and the pluripotent stem cell can be made into a single cell. Further, the detachment of the undifferentiated cells from the substrate can be promoted by the step [1]. If step [1] is not performed, pluripotent stem cells cannot be detached from a single cell.

In the step [1], an intercellular binding dissociation solution containing trypsin is used. Here, in the present invention, “trypsin” refers to “EC.3.4.21.4” of a protease that is secreted from the pancreas or a protease that has properties equivalent to those of the protease. Examples of commercially available products include Trypsin (trademark), TrypLE (trademark) SELECT, and TrypLE (trademark) EXPRES (all manufactured by Thermo Fisher Scientific). In the present invention, the term "intercellular bond dissociation solution" refers to a solution having an action of dissociating a bond (intercellular bond) formed between cells through a transmembrane protein or the like. The intercellular binding dissociation solution may contain a chelating agent and the like in addition to trypsin, an enzyme having the same proteolytic action as trypsin.
By using the intercellular binding dissociation solution containing trypsin, pluripotent stem cells can be detached from a single cell. Without trypsin, single cells cannot exfoliate pluripotent stem cells. Since it is suitable for exfoliating pluripotent stem cells with a single cell and further increasing the survival rate of the cells, the trypsin content is preferably 0.01 to 2.5 wt%, and 0.1 to 1 wt%. More preferably, 0.2 to 0.5 wt% is particularly preferable, and 0.25 to 0.35 wt% is most preferable.

Since the intercellular binding dissociation solution is suitable for making cells into single cells, it is preferable to further contain a chelating agent of 5 × 10 ^{−10 to} 5 × 10 ⁻¹ mol / L, 5 × 10 ⁻⁶ to 1 × 10 ⁻¹ mol / L is more preferable, 5 × 10 ^{−4 to} 5 × 10 ⁻² mol / L is particularly preferable, and 1 × 10 ^{−3 to} 5 × 10 ⁻² mol / L. Is most preferred.

  The chelating agent is not particularly limited except that it is a chelating agent. For example, ethylenediaminetetraacetic acid, ethylenediamine, ethylenediaminetetramethylenephosphonic acid, glycoletherdiaminetetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, N- (2 -Hydroxyethyl) ethylenediamine-N, N ', N'-triacetic acid, triethylenetetramine-N, N, N', N ", N '", N' "-hexaacetic acid, 1,3-propane Diamine-N, N, N ', N'-tetraacetic acid, 1,3-diamino-2-propanol-N, N, N', N'-tetraacetic acid, N- (2-hydroxyethyl) iminodiacetic acid, N, N-di (2-hydroxyethyl) glycine, glycol ether diaminetetraacetic acid, dicarboxymethylglutamic acid, ethylenedia -N, N'-disuccinic acid, 2,3-dihydroxybenzoic acid, iminodiacetic acid, etidronic acid, citric acid, mugineic acid, bipyridine, porphyrin, phenanthroline, porphyrin, crown ether, cyclen, 18-crown-6, Deferoxamine, daughter octreotate, nicotianamine, dimercaprol, siderophore and the like can be mentioned. A chelating agent that forms a chelate with a metal ion of 1 to 4 valences is preferable because it is suitable for making the cells into a single cell and is also suitable for enhancing the viability of the cells. Chelating agents that form chelates with ions are more preferred, chelating agents that form chelates with calcium ions are particularly preferred, and ethylenediaminetetraacetic acid is most preferred.

  The contact time between the pluripotent stem cells and the intercellular binding dissociation solution in the above step [1] is preferably 1 since the pluripotent stem cells are single cells and are suitable for increasing the survival rate of the cells. The time is preferably from 30 seconds to 30 minutes, more preferably from 10 seconds to 10 minutes, particularly preferably from 1 minute to 5 minutes, most preferably from 2 minutes to 4 minutes.

  The temperature of the intercellular binding dissociation solution in the step [1] is not particularly limited, but is preferably 10 to 40 ° C, more preferably 20 to 40 ° C, because it is suitable for activating trypsin. -40 ° C is particularly preferred, and 37 ° C is most preferred.

  After the step [1], the pluripotent stem cells are in a state of being adhered to the substrate. The survival rate of the pluripotent stem cells can be increased by allowing the pluripotent stem cells to adhere to the substrate after the step [1]. [1] When the pluripotent stem cells are detached from the substrate during the process, the survival rate of the pluripotent stem cells is reduced.

  In the step [2] of the method for detaching pluripotent stem cells of the present invention, the pluripotent stem cells are washed to remove trypsin while the pluripotent stem cells remain adhered to the substrate. The method for washing the pluripotent stem cells and removing trypsin is not particularly limited.For example, after aspirating and removing the intercellular binding dissociated solution that has been brought into contact with the pluripotent stem cells, The pluripotent stem cells can be washed by contacting the pluripotent stem cells and then removing the phosphate buffered saline. In order to increase the survival rate of the pluripotent stem cells, the pluripotent stem cells are in a state of being adhered to the substrate after the step [2] in the method for detaching the pluripotent stem cells of the present invention. [2] When the pluripotent stem cells are detached from the substrate during the process, the survival rate of the pluripotent stem cells is reduced.

  After the step [2], if necessary, removal of the intercellular binding dissociation solution or replacement with a medium may be performed. The medium is not particularly limited, but is suitable for maintaining undifferentiated pluripotent stem cells, for example, known as a factor for maintaining undifferentiated pluripotent stem cells, A medium to which one or more of insulin, transferrin, selenium, ascorbic acid, sodium bicarbonate, basic fibroblast growth factor, transforming growth factor β (TGFβ), CCL2, activin, and 2-mercaptomethanol are preferably added. It is preferable to use a medium containing insulin, transferrin, selenium, ascorbic acid, sodium bicarbonate, basic fibroblast growth factor, transforming growth factor β (TGFβ), and more preferably basic fibroblast growth factor. An added medium is particularly preferred.

  The type of medium to which the basic fibroblast growth factor has been added is not particularly limited. For example, commercially available products include DMEM (manufactured by Sigma-Aldrich Co. LLC) and Ham's F12 (Sigma-Aldrich Co. LLC). D-MEM / Ham's F12 (Sigma-Aldrich Co. LLC), Primate ES Cell Medium (Reprocell), StemFit AK02N (Ajinomoto Co.), StemFit AK03 (Ajinomoto) MTeSR1 (manufactured by STEMCELL TECHNOLOGIES), TeSR-E8 (manufactured by STEMCELL TECHNOLOGIES), ReproNaive (manufactured by REPROCELL), ReproXF (manufactured by REPR) CELL), ReproFF (manufactured by REPROCELL), ReproFF2 (manufactured by REPROCELL), NutriStem (manufactured by Biologic Industries), iSTEM (manufactured by Takara Bio Inc.), GS2-M (manufactured by Takara Bio Inc.) Co., Ltd.) and hPSC Growth Medium DXF (manufactured by PromoCell). Primate ES Cell Medium (manufactured by REPROCELL), StemFit AK02N (manufactured by Ajinomoto Co.) or StemFit AK03 (manufactured by Ajinomoto Co.) is suitable for maintaining the undifferentiated state of pluripotent stem cells. StemFit AK02N (manufactured by Ajinomoto Co.) or StemFit AK03 (manufactured by Ajinomoto Co.) is more preferable, and StemFit AK02N (manufactured by Ajinomoto Co.) is particularly preferable.

Further, since it is suitable for enhancing the survival rate of cells, a Rho-binding kinase inhibitor may be added as necessary. Particularly when human pluripotent stem cells are used, examples of the Rho-binding kinase inhibitor include (R)-(+)-trans-N- (4-pyridyl) -4- (1-aminoethyl)- Cyclohexanecarboxamide.2HCl.H2O (Y-27632 manufactured by Wako Pure Chemical Industries, Ltd.), 1- (5-isoquinolinesulfonyl) homopiperazine Hydrochloride (HA1077 manufactured by Wako Pure Chemical Industries, Ltd.) can be used. The concentration of the Rho-binding kinase inhibitor added to the medium is in a range that is effective for maintaining the survival of human pluripotent stem cells and does not affect the undifferentiated state of human pluripotent stem cells, 1 × 10 ^{−6 to} 5 × 10 ⁻⁵ mol / L is preferable, 3 × 10 ⁻⁶ to 2 × 10 ⁻⁵ mol / L is more preferable, and 8 × 10 ^{−6 to} 1.2 × 10 ⁻⁵ mol / L is preferable. L is particularly preferred.
In the step [3] of the method for exfoliating pluripotent stem cells of the present invention, the substrate is cooled and the pluripotent stem cells are exfoliated from the substrate. It is preferable to cool at a temperature of 0 to 30 ° C. for 1 second to 60 minutes, and more preferably to cool at a temperature of 2 to 25 ° C. for 1 second to 60 minutes, since it is suitable for increasing the viability of cells. It is particularly preferable to cool at a temperature of 2 to 20 ° C for 1 second to 60 minutes, most preferably at a temperature of 2 to 10 ° C for 1 second to 60 minutes.

  In the method for detaching pluripotent stem cells of the present invention, a substrate having a layer made of a temperature-responsive polymer is used. When the substrate has a layer made of a temperature-responsive polymer, the pluripotent stem cells can be detached by cooling the substrate in the step [3]. If there is no layer made of a temperature-responsive polymer, the pluripotent stem cells cannot be detached in step [3].

  In the present invention, the product of the layer thickness of the layer made of the temperature-responsive polymer and the content ratio of the temperature-responsive component is 100 nm or less. When the product of the layer thickness of the layer made of the temperature-responsive polymer and the content ratio of the temperature-responsive component is 100 nm or less, the pluripotent stem cells can be firmly adhered to the base material. In step 2], the cells can be prevented from detaching. If the product of the layer thickness of the temperature-responsive polymer and the content ratio of the temperature-responsive component exceeds 100 nm, the cells are likely to be detached in the steps [1] and [2] and the cell survival rate is reduced. Becomes In the above [1] and [2] steps, the product of layer thickness × temperature-responsive component is preferably 50 nm or less because it is suitable for suppressing cell detachment and increasing cell survival rate. Preferably, it is particularly preferably 40 nm or less, most preferably 20 nm or less. Here, in the present invention, the “layer thickness” of the layer made of the temperature-responsive polymer refers to the length of the layer made of the temperature-responsive polymer from the interface closest to the substrate to the interface closest to the surface in the direction outside the substrate surface. In the range where the layer thickness exceeds 10 nm, a cross-sectional image is measured by a transmission electron microscope using an ultrathin section of the culture substrate prepared by a microtome, and the distance of 10 points selected at random is measured. , Can be calculated by averaging. When the layer thickness is in the range of 10 nm or less, it can be measured using an ellipsometer. Further, in the present invention, the “content ratio of the temperature-responsive component” indicates the weight ratio of the temperature-responsive monomer unit contained in the temperature-responsive polymer.

  In addition, since the pluripotent stem cells are suitable for exfoliating the pluripotent stem cells in a short time in the step [3] and reducing damage to the cells, the product of the layer thickness × the content ratio of the temperature-responsive component is 5 nm or more. Preferably, it is 10 nm or more, more preferably 20 nm or more, and most preferably 30 nm or more.

  Examples of the base material having a layer made of the temperature-responsive polymer include a base material having a layer made of a temperature-responsive polymer having a response temperature to water in the range of 0 ° C to 50 ° C on the surface. The response temperature is preferably in the range of 0 ° C. to 50 ° C., more preferably 5 ° C. to 35 ° C., and particularly preferably 10 ° C. to 25 ° C. because it is suitable for increasing the survival rate of pluripotent stem cells. Preferably, 15 ° C to 25 ° C is most preferred.

  The monomer unit constituting the temperature-responsive polymer is not particularly limited, and examples thereof include (meth) acrylamide compounds such as acrylamide and methacrylamide; N, N-diethylacrylamide, N-ethylacrylamide, and Nn -Propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N N-alkyl-substituted (meth) acrylamide derivatives such as -tetrahydrofurfurylacrylamide and N-tetrahydrofurfuryl methacrylamide; N, N-dimethyl (meth) acrylamide, N, N-ethylmethyla N, N-dialkyl-substituted (meth) acrylamide derivatives such as rilamide and N, N-diethylacrylamide; 1- (1-oxo-2-propenyl) -pyrrolidine, 1- (1-oxo-2-propenyl) -piperidine, 4- (1-oxo-2-propenyl) -morpholine, 1- (1-oxo-2-methyl-2-propenyl) -pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) -piperidine, (Meth) acrylamide derivatives having a cyclic group such as 4- (1-oxo-2-methyl-2-propenyl) -morpholine; vinyl ethers such as methyl vinyl ether; N, N-diethylacrylamide, Nn-propylacrylamide, N-isopropylacryl Amide, Nn-propyl methacrylamide, N-ethoxyethyl acrylamide, N-tetrahydrofurfuryl acrylamide, N-tetrahydrofurfuryl methacrylamide is preferred, N, N-diethylacrylamide, N-isopropylacrylamide is more preferred, and N- Isopropylacrylamide is particularly preferred.

  The method of forming the layer using the temperature-responsive polymer is not particularly limited. However, since it is suitable for firmly fixing the temperature-responsive polymer to the substrate, the monomer of the temperature-responsive polymer is preferably used. Method of electron beam polymerization of units on a substrate, method of immobilizing on a substrate by heat or light using a thermo- or photo-reactive temperature-responsive polymer, monomer units of a temperature-responsive polymer and others A method using a copolymer with the above monomer unit can be suitably used.

  In the method using a copolymer of the monomer unit of the temperature-responsive polymer and other monomer units, since it is suitable for firmly immobilizing the copolymer to the substrate, at least It preferably contains a water-insoluble polymer monomer unit. Further, since the structure of the copolymer is suitable for controlling the response temperature of the temperature-responsive polymer, the structure of the copolymer is preferably a block copolymer.

  The monomer unit of the polymer insoluble in water is not particularly limited, and examples thereof include styrene, 2-hydroxyphenyl acrylate, 2-hydroxyphenyl methacrylate, 3-hydroxyphenyl acrylate, 3-hydroxyphenyl methacrylate, Hydroxyphenyl acrylate, 4-hydroxyphenyl methacrylate, N- (2-hydroxyphenyl) acrylamide, N- (2-hydroxyphenyl) methacrylamide, N- (3-hydroxyphenyl) acrylamide, N- (3-hydroxyphenyl) methacryl Amide, N- (4-hydroxyphenyl) acrylamide, N- (4-hydroxyphenyl) methacrylamide, n-butyl acrylate, n-butyl methacrylate, isobutyl acrylate, isobutyl methacrylate Acrylate, t-butyl acrylate, t-butyl methacrylate, n-hexyl acrylate, n-hexyl methacrylate, n-octyl acrylate, n-octyl methacrylate, n-decyl acrylate, n-decyl methacrylate, n-dodecyl acrylate, n-dodecyl Examples include methacrylate, n-tetradecyl acrylate, and n-tetradecyl methacrylate.

  The structural unit ratio of the temperature-responsive polymer in the copolymer is preferably 5 mol% or more, more preferably 20 mol% or more, particularly preferably 30 mol% or more, and more preferably 40 mol%, from the viewpoint of enhancing the cooling releasability of the pluripotent stem cells. % Or more is most preferable. Further, since it is suitable for firmly fixing the copolymer to the base material, the structural unit ratio of the temperature-responsive polymer is preferably 95 mol% or less, more preferably 90 mol% or less. , 85 mol% or less, and most preferably, 80 mol% or less.

  The molecular weight of the temperature-responsive polymer is not particularly limited, but is preferably from 1,000 to 1,000,000 in number average molecular weight, because it is suitable for increasing the strength of the layer made of the temperature-responsive polymer. It is more preferably from 500,000 to 500,000, particularly preferably from 5000 to 300,000, and most preferably from 10,000 to 200,000.

  The material of the base material in the present invention is not particularly limited, but not only materials such as glass and polystyrene that are usually used for cell culture, but also materials that can be generally given a form, for example, polycarbonate and polyethylene terephthalate. And polymer compounds such as polyvinylidene fluoride, polytetrafluoroethylene, polyethylene, polypropylene, and polymethyl methacrylate; ceramics; and metals. From the viewpoint of easiness of the culturing operation, the material of the substrate preferably contains at least one of glass, polystyrene, polycarbonate, polyethylene terephthalate, polyvinylidene fluoride, polytetrafluoroethylene, polyethylene, and polypropylene, and glass, polystyrene, and polycarbonate. And more preferably at least one of polyethylene terephthalate, and polystyrene, polycarbonate and polyethylene terephthalate are particularly preferred because they are suitable for enhancing flexibility.

  The shape of the substrate in the present invention is not particularly limited, and may be a planar shape such as a plate or a film, or may be a fiber, a porous particle, a porous membrane, or a hollow fiber. Further, a container (cell culture dish such as a Petri dish, a flask, a plate, or the like) generally used for cell culture or the like may be used. From the viewpoint of easiness of the culturing operation, a porous membrane having a planar shape such as a plate or a film or a flat membrane is preferable.

Hereinafter, the present invention will be described in detail with reference to embodiments for carrying out the present invention. However, this is an exemplification for describing the present invention, and is not intended to limit the present invention to the following contents. Further, the present invention can be appropriately modified and implemented within the scope of the present invention. Unless otherwise specified, commercially available reagents were used.
<Polymer composition>
Nuclear magnetic resonance measurement apparatus (JEOL Ltd., trade name JNM-GSX400) Proton nuclear magnetic resonance spectroscopy ⁽¹ H-NMR) spectrum analysis using, or nuclear magnetic resonance measuring device (Bruker, trade name AVANCEIIIHD500) Was determined by carbon nuclear magnetic resonance spectroscopy ( ^13C -NMR) spectrum analysis using
<Molecular weight and molecular weight distribution of polymer>
The weight average molecular weight (Mw), number average molecular weight (Mn) and molecular weight distribution (Mw / Mn) were measured by gel permeation chromatography (GPC). The GPC apparatus uses HLC-8320GPC manufactured by Tosoh Corporation, the column uses two TSKgel Super AWM-H manufactured by Tosoh Corporation, the column temperature is set to 40 ° C., and the eluent contains 10 mM sodium trifluoroacetate. The measurement was performed using N, N-dimethylformamide containing 1,1,1,3,3,3-hexafluoro-2-isopropanol or 10 mM lithium bromide. The measurement sample was prepared at 1.0 mg / mL and measured. For the calibration curve of the molecular weight, polymethyl methacrylate (manufactured by Polymer Laboratories) having a known molecular weight was used.

[Example 1]
As a substrate, a polystyrene dish (manufactured by Corning Incorporated) having a layer formed of a temperature-responsive polymer was used, and human iPS cells 201B7 were seeded at a density of 1300 cells / cm ² at 37 ° C. and a CO ₂ concentration of 5 %. The medium used was StemFitAK02N (manufactured by Ajinomoto Co., Inc.). Until 24 hours after the cell seeding, Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration: 10 μM) and iMatrix-511 solution (manufactured by Nippi Co., Ltd.) (2. (5 μL / mL).

  At 24 hours, 96 hours, and 144 hours after the cell seeding, the state of the cells was observed with a phase contrast microscope. In each case, cell adhesion and proliferation were confirmed, and the medium was replaced with a new medium. After the medium was exchanged 144 hours after the cell seeding, it was confirmed that the cells were present in a colony on the substrate.

After removing the medium and washing with phosphate buffered saline, cells obtained by adding 0.5 × TrypLE SELECT and ethylenediaminetetraacetic acid to phosphate buffered saline to a concentration of 5 × 10 ⁻³ mol / L. The bond separation liquid was added to the dish and treated at 37 ° C. for 3 minutes. Aspirate and remove the intercellular binding dissociation solution, add phosphate buffered saline, and remove the phosphate buffered saline by suction. After washing the cells with the substrate adhered, add Y-27632. The prepared medium was added to the dish. The cells were detached by cooling at 15 ° C. for 10 minutes. Cell viability was 96.5%.

  The synthesis and coating of the temperature-responsive polymer were performed by the following methods.

  In a test tube, 0.40 g (0.1 mmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl) sulfanyl] pentanoic acid, 7.11 g (50 mmol) of n-butyl methacrylate, azobis (isobutyronitrile) 33 mg (0.2 mmol) was added and dissolved in 50 mL of 1,4-dioxane. After deaeration by nitrogen bubbling for 30 minutes, the reaction was carried out at 70 ° C. for 24 hours. After completion of the reaction, the reaction solvent was distilled off under reduced pressure using a rotary evaporator, and the reaction solution was concentrated. The concentrated solution was poured into 250 mL of methanol, and the precipitated yellow oily substance was recovered and dried under reduced pressure to obtain an n-butyl methacrylate polymer.

  0.9 g (0.3 mmol) of the n-butyl methacrylate polymer, 8.14 g (72 mmol) of N-isopropylacrylamide, and 5 mg (0.03 mmol) of azobisisobutyronitrile were added to a test tube, and 1,4- It was dissolved in 15 mL of dioxane. After deaeration by nitrogen bubbling for 30 minutes, the reaction was carried out at 65 ° C. for 17 hours. After the completion of the reaction, the reaction solvent was diluted with acetone, poured into 500 mL of hexane, and the precipitated pale yellow solid was recovered and dried under reduced pressure to obtain a copolymer of N-isopropylacrylamide and n-butyl methacrylate.

  A 0.5 wt% ethanol solution of a copolymer of N-isopropylacrylamide and n-butyl methacrylate was spin-coated at 2,000 rpm on a polystyrene dish to coat a temperature-responsive polymer.

  The temperature-responsive polymer had a response temperature of 32 ° C., a constitutional unit ratio of 4 mol% of n-butyl methacrylate, 96 mol% of N-isopropylacrylamide, and a molecular weight of Mn 70,000. The thickness of the coating of the temperature-responsive polymer on the polystyrene dish was 25 nm.

[Example 2]
Using the base material prepared by the following method in Example 1, after performing cooling at 15 ° C. for 10 minutes, performing an operation of hitting the base material 20 times to apply vibration, and otherwise exfoliating the cells in the same manner as in Example 1. did. Cell viability was 92.3%.

  A 0.1 wt% ethanol solution of a copolymer of N-isopropylacrylamide and n-butyl methacrylate was spin-coated on a polystyrene dish at 2000 rpm to coat a temperature-responsive polymer. The thickness of the coating of the temperature-responsive polymer on the polystyrene dish was 5 nm.

[Comparative Example 1]
Example 1 was repeated except that a layer made of a temperature-responsive polymer was not used and a polystyrene dish was used as it was. The cells did not detach from the substrate and could not be recovered.

[Comparative Example 2]
The cells were peeled off in the same manner as in Example 1 except that the substrate prepared in Example 1 was used in the following manner. Almost all cells were detached from the substrate while the pluripotent stem cells were in contact with the cell-binding dissociation solution, and the viability was 0%.

  A 2 wt% ethanol solution of a copolymer of N-isopropylacrylamide and n-butyl methacrylate was spin-coated at 2,000 rpm on a polystyrene dish to coat a temperature-responsive polymer. The thickness of the coating of the temperature-responsive polymer on the polystyrene dish was 120 nm.

[Comparative Example 3]
The procedure of Example 1 was repeated except that the operation of contacting the pluripotent stem cells with the intercellular binding dissociation liquid was not performed in Example 1. The cells detached only partially from the substrate, and the detached cells were clumps of cells, and single cells could not be recovered.

[Comparative Example 4]
In Example 1, the cell-bound dissociation solution that had been brought into contact with the pluripotent stem cells was not removed by suction, and a cooling-peeling operation was performed with trypsin remaining, to detach the cells from the substrate. The cells detached, but most of the cells died, and the viability was 29.5%.

Claims (3)

A method for exfoliating pluripotent stem cells cultured on a substrate, wherein the substrate has a layer of a temperature-responsive polymer having a product of a layer thickness and a content ratio of a temperature-responsive polymer component of 100 nm or less, A method for exfoliating pluripotent stem cells, comprising the following steps [1] to [3].
[1] A step of contacting a pluripotent stem cell cultured on a substrate with an intercellular binding dissociation solution containing trypsin.
[2] a step of removing the trypsin-containing cell-binding dissociation solution while the pluripotent stem cells remain adhered to the substrate.
[3] A step of cooling the substrate and peeling the pluripotent stem cells from the substrate. 2. The method according to claim 1, wherein the contact time between the trypsin-containing intercellular binding dissociation solution and the pluripotent stem cells adhered to the substrate in the step [1] is 1 second to 5 minutes. A method for detaching competent stem cells. The method according to claim 1 or 2, wherein the pluripotent stem cells are induced pluripotent stem cells.