JP7143667B2 - Method for exfoliating pluripotent stem cells - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for detaching pluripotent stem cells, which is excellent in mass productivity, detaches pluripotent stem cells with a high survival rate, and can be collected as single cells.

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

Conventionally, to recover pluripotent stem cells cultured on a substrate as single cells, the pluripotent stem cells are detached from the substrate by scraping the substrate with a cell scraper after treating with an enzyme such as trypsin. I was letting However, in such a method of exfoliating pluripotent stem cells, it is necessary to scrape the entire substrate with a cell scraper, which makes it difficult to recover a large amount of cells using a large-area substrate.

As another method for detaching cells without using a cell scraper, a method using a temperature-responsive substrate 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, and cells are exfoliated by temperature change to recover a cell sheet. A method for doing so is disclosed. However, the method described in Patent Document 1 is mainly aimed at collecting cell sheets, and cannot obtain single-cell pluripotent stem cells.

JP 2009-131275 A

An object of the present invention is to provide a method for detaching pluripotent stem cells that is excellent in mass productivity, detaches pluripotent stem cells with a high survival rate, and can be collected as single cells.

In view of the above points, the present inventors have made intensive studies, and as a result, using a substrate having a layer of a temperature-responsive polymer in which the product of the layer thickness x the content ratio of the temperature-responsive component is 100 nm or less, trypsin The inventors have found that the above problems can be solved by a step of contacting pluripotent stem cells with an intercellular junction dissociation solution containing, and have completed the present invention.

That is, the present invention provides a method for exfoliating pluripotent stem cells cultured on a base material, wherein the base material includes a layer of a temperature-responsive polymer having a product of layer thickness x temperature-responsive component content ratio of 100 nm or less. A method for exfoliating pluripotent stem cells, characterized by comprising the following steps [1] to [3].
[1] A step of contacting an intercellular junction dissociation solution containing trypsin with pluripotent stem cells cultured on a substrate.
[2] A step of removing the intercellular junction dissociation solution containing trypsin while the pluripotent stem cells remain adhered to the substrate.
[3] A step of cooling the substrate and exfoliating the pluripotent stem cells from the substrate.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a method for detaching pluripotent stem cells that is excellent in mass productivity and capable of detaching pluripotent stem cells as single cells.

EMBODIMENT OF THE INVENTION Hereinafter, the form (only henceforth "this Embodiment") for implementing this invention is demonstrated in detail. The following embodiments are examples for explaining the present invention, and are not intended to limit the present invention to the following contents. The present invention can be appropriately modified and implemented within the scope of its gist.

As used herein, the term "temperature-responsive polymer" refers to a polymer whose degree of hydrophilicity/hydrophobicity changes with temperature changes. In addition, when the temperature of the polymer is increased from a low temperature to a high temperature in the presence of water molecules, the degree of hydrophilicity/hydrophobicity of the polymer changes to become more hydrophobic at a certain temperature. There is a temperature-responsive polymer that changes from water-insoluble to water-insoluble, and this boundary temperature is called "Lower Critical Solution Temperature (LCST)". In general, when a temperature-responsive polymer dissolves in water at a temperature below the LCST, it becomes insoluble in water at a temperature above the LCST. When the temperature-responsive polymer is water-insoluble, it does not have an LCST, but has a response temperature at which the degree of hydrophilicity/hydrophobicity changes with temperature changes.

In the present invention, the type of pluripotent stem cells is not particularly limited, but for example, pulmonary stem cells (ES cells), induced pluripotent stem cells (iPS cells), and nuclear transplanted ES cells (ntES cells) are used. iPS cells are particularly preferred. In addition, although the animal from which the pluripotent stem cells are derived is not particularly limited, it can be derived from mammals, for example. Examples of mammals include rodents (mice, rats, etc.) and primates (monkeys, humans, etc.), and may be laboratory animals or companion animals. In the present invention, it is preferably derived from primates, particularly preferably human.

The step [1] in the method for exfoliating pluripotent stem cells of the present invention is to bring the pluripotent stem cells into contact with the intercellular junction dissociation fluid. By bringing the pluripotent stem cells into contact with the intercellular junction dissociation solution, the cell-cell junctions of the pluripotent stem cells can be dissociated and the pluripotent stem cells can be converted into single cells. In addition, the step [1] can promote detachment of undifferentiated cells from the substrate. [1] If the step is not performed, pluripotent stem cells cannot be exfoliated with single cells.

In the step [1], an intercellular junction dissociation solution containing trypsin is used. Here, "trypsin" in the present invention indicates "EC.3.4.21.4" which is a proteolytic enzyme secreted from the pancreas, or a degrading enzyme having properties equivalent to said proteolytic enzyme. 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 junction dissociation solution" refers to a solution that has the effect of dissociating the bonds (intercellular bonds) formed between cells via a transmembrane protein or the like. The intercellular junction dissociation solution may contain trypsin, an enzyme having the same proteolytic activity as trypsin, and a chelating agent.
By using an intercellular junction dissociation solution containing trypsin, single cell pluripotent stem cells can be detached. Without trypsin, single cells cannot detach pluripotent stem cells. The content of trypsin is preferably 0.01 to 2.5 wt%, preferably 0.1 to 1 wt%, because it is suitable for exfoliating pluripotent stem cells with single cells and increasing cell viability. More preferably, 0.2 to 0.5 wt% is particularly preferred, and 0.25 to 0.35 wt% is most preferred.

The intercellular junction dissociation solution preferably further contains 5×10 ⁻¹⁰ to 5×10 ⁻¹ mol/L of a chelating agent, since it is suitable for converting cells into single cells. 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 more preferable. is most preferred.

The chelating agent is not particularly limited except that it is a chelating agent. -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 diamine tetraacetic acid, dicarboxymethylglutamic acid, ethylenediamine-N,N'-disuccinic acid, 2,3-dihydroxybenzoic acid, iminodiacetic acid, etidronic acid, citric acid acid, mugineic acid, bipyridine, porphyrin, phenanthroline, porphyrin, crown ether, cyclen, 18-crown-6, deferoxamine, dotaoctreotate, nicotianamine, dimercaprol, siderophore and the like. A chelating agent that forms a chelate with a monovalent to tetravalent metal ion is preferable because it is suitable for making cells into single cells and is suitable for increasing the viability of cells, and divalent metals 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 junction dissociation solution in the step [1] is suitable for converting the pluripotent stem cells into single cells and increasing the survival rate of the cells. Seconds to 30 minutes are preferred, 10 seconds to 10 minutes are more preferred, 1 minute to 5 minutes are particularly preferred, and 2 to 4 minutes are most preferred.

The temperature of the intercellular junction dissociation solution in the step [1] is not particularly limited, but is preferably 10 to 40°C, more preferably 20 to 40°C, since 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 adhering to the substrate. The survival rate of the pluripotent stem cells can be increased by keeping the pluripotent stem cells adhered to the substrate after the step [1]. [1] If the pluripotent stem cells are detached from the substrate during the process, it causes a decrease in the viability of the pluripotent stem cells.

In step [2] in 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 pluripotent stem cells and removing trypsin is not particularly limited. The pluripotent stem cells can be washed by contacting the potent stem cells and then removing the phosphate buffered saline. In order to increase the survival rate of pluripotent stem cells, the pluripotent stem cells are in a state of adhering to the substrate after step [2] in the method of detaching pluripotent stem cells of the present invention. [2] If the pluripotent stem cells are detached from the substrate during the process, it causes a decrease in the viability of the pluripotent stem cells.

After the step [2], the intercellular junction dissociation solution may be removed or replaced with a medium, if necessary. Although the medium is not particularly limited, it is suitable for maintaining the undifferentiated state of pluripotent stem cells. Preferably medium supplemented with one or more of insulin, transferrin, selenium, ascorbic acid, sodium bicarbonate, basic fibroblast growth factor, transforming growth factor beta (TGFβ), CCL2, activin, 2-mercaptomethanol Preferably, media containing insulin, transferrin, selenium, ascorbic acid, sodium bicarbonate, basic fibroblast growth factor, transforming growth factor beta (TGFβ) are more preferred, and basic fibroblast growth factor is preferred. Supplemented media are particularly preferred.

The type of medium to which the basic fibroblast growth factor is added is not particularly limited. manufactured by), D-MEM/Ham's F12 (manufactured by Sigma-Aldrich Co. LLC), Primate ES Cell Medium (manufactured by REPROCELL Co., Ltd.), StemFit AK02N (manufactured by Ajinomoto Co., Inc.), StemFit AK03 (manufactured by Ajinomoto Co., Inc. (manufactured by STEMCELL TECHNOLOGIES), mTeSR1 (manufactured by STEMCELL TECHNOLOGIES), TeSR-E8 (manufactured by STEMCELL TECHNOLOGIES), ReproNaive (manufactured by REPROCELL), ReproXF (manufactured by REPROCELL), ReproFF (manufactured by REPROCELL), ReproFF2 ( REPROCELL Co., Ltd.), NutriStem (Biological Industries Inc.), iSTEM (Takara Bio Inc.), GS2-M (Takara Bio Inc.), hPSC Growth Medium DXF (PromoCell), etc. can be mentioned. Primate ES Cell Medium (manufactured by REPROCELL Co., Ltd.), StemFit AK02N (manufactured by Ajinomoto Co., Inc.) or StemFit AK03 (manufactured by Ajinomoto Co., Inc.) are suitable for maintaining the undifferentiated state of pluripotent stem cells. is preferred, StemFit AK02N (manufactured by Ajinomoto Co.) or StemFit AK03 (manufactured by Ajinomoto Co.) is more preferred, and StemFit AK02N (manufactured by Ajinomoto Co.) is particularly preferred.

In addition, a Rho-binding kinase inhibitor may be added as necessary, as it is suitable for increasing cell viability. Especially when using human pluripotent stem cells, examples of Rho-binding kinase inhibitors include (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)- Cyclohexanecarboxamide.2HCl.H2O (Y-27632, manufactured by Wako Pure Chemical Industries, Ltd.) and 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 within 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 more preferable. L is particularly preferred.
In step [3] in the method for detaching pluripotent stem cells of the present invention, the substrate is cooled and the pluripotent stem cells are detached from the substrate. It is preferable to cool at a temperature of 0 to 30° C. for 1 second to 60 minutes, more preferably at a temperature of 2 to 25° C. for 1 second to 60 minutes, because it is suitable for increasing the viability of cells. Cooling at a temperature of 2-20° C. for 1 second to 60 minutes is particularly preferred, and cooling at a temperature of 2-10° C. for 1 second to 60 minutes is most preferred.

In the method of exfoliating pluripotent stem cells of the present invention, a base material having a layer of a temperature-responsive polymer is used. Since the base material has a layer of the temperature-responsive polymer, the pluripotent stem cells can be exfoliated by cooling the base material in step [3]. If there is no layer of temperature-responsive polymer, the pluripotent stem cells cannot be exfoliated in step [3].

In the present invention, the product of the layer thickness of the layer 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 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 strongly adhered to the substrate, and the above [1] and [ 2] It is possible to suppress detachment of cells in the step. When the product of the layer thickness of the layer of the temperature-responsive polymer and the content ratio of the temperature-responsive component exceeds 100 nm, the cells tend to detach in steps [1] and [2], and the cell viability decreases. becomes. It is preferable that the product of layer thickness x temperature-responsive component is 50 nm or less because it is suitable for suppressing cell detachment in the steps [1] and [2] and increasing the survival rate of cells. It is preferably 40 nm or less, particularly preferably 40 nm or less, and most preferably 20 nm or less. Here, in the present invention, the "layer thickness" of the layer made of the temperature-responsive polymer means the length of the layer made of the temperature-responsive polymer in the substrate out-of-plane direction from the interface closest to the substrate to the interface closest to the surface. In the range where the layer thickness exceeds 10 nm, an ultra-thin section of the culture substrate prepared by microtome is used to measure the cross-sectional image with a transmission electron microscope, and the distance is measured at 10 randomly selected points. , can be calculated by averaging. In addition, 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 rate of the temperature-responsive component" indicates the weight rate of the temperature-responsive monomer unit contained in the temperature-responsive polymer.

In addition, since it is 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. 10 nm or more is more preferable, 20 nm or more is particularly preferable, and 30 nm or more is most preferable.

Examples of the substrate having a layer made of the temperature-responsive polymer include substrates having a layer made of the 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., particularly 10° C. to 25° C. Preferably, 15°C to 25°C is most preferred.

The monomer unit constituting the thermoresponsive polymer is not particularly limited, but examples include (meth)acrylamide compounds such as acrylamide and methacrylamide; N,N-diethylacrylamide, N-ethylacrylamide, 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 tetrahydrofurfuryl acrylamide and N-tetrahydrofurfuryl methacrylamide; N,N-dialkyl-substituted (meth)acrylamide derivatives; 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, 4-(1-oxo-2-methyl -2-propenyl)-(meth)acrylamide derivatives having a cyclic group such as morpholine; and vinyl ethers such as methyl vinyl ether. - diethylacrylamide, Nn-propylacrylamide, N-isopropylacrylamide, Nn-propylmethacrylamide, N-ethoxyethylacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylamide are preferred, N,N -Diethylacrylamide and N-isopropylacrylamide are more preferred, and N-isopropylacrylamide is particularly preferred.

The method for forming the layer of the temperature-responsive polymer is not particularly limited, but since it is suitable for firmly immobilizing the temperature-responsive polymer on the substrate, the monomer of the temperature-responsive polymer A method of electron beam polymerization of a unit on a substrate, a method of immobilizing a thermally or photoreactive temperature responsive polymer on a substrate by heat or light, a monomer unit of a temperature responsive polymer, and others. A method using a copolymer with a monomer unit of can be preferably used.

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

The monomer units of the water-insoluble polymer are not particularly limited, but examples include styrene, 2-hydroxyphenyl acrylate, 2-hydroxyphenyl methacrylate, 3-hydroxyphenyl acrylate, 3-hydroxyphenyl methacrylate, 4- 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, 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 methacrylate, n-tetradecyl acrylate, n-tetradecyl methacrylate can be mentioned.

From the viewpoint of enhancing the cooling exfoliation property of pluripotent stem cells, 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 40 mol. % or more is most preferable. Moreover, since it is suitable for firmly fixing the copolymer to the substrate, the structural unit ratio of the temperature-responsive polymer is preferably 95 mol % or less, more preferably 90 mol % or less. , is particularly preferably 85 mol % or less, most preferably 80 mol % or less.

The molecular weight of the temperature-responsive polymer is not particularly limited, but the number-average molecular weight is preferably 1,000 to 1,000,000, preferably 2,000, because it is suitable for increasing the strength of the layer 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 substrate in the present invention is not particularly limited, but it is not limited to materials such as glass and polystyrene that are usually used for cell culture, but also materials that can generally be shaped, such as polycarbonate and polyethylene terephthalate. , polyvinylidene fluoride, polytetrafluoroethylene, polyethylene, polypropylene, polymethyl methacrylate, ceramics, and metals. From the viewpoint of facilitating culture operations, 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 polyethylene terephthalate, and polystyrene, polycarbonate, and polyethylene terephthalate are particularly preferred because they are suitable for increasing 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 film, or may be a fiber, porous particles, porous membrane, or hollow fiber. In addition, a container generally used for cell culture or the like (cell culture dish such as Petri dish, flask, plate, etc.) may also be used. From the viewpoint of easiness of culturing operation, it is preferably a planar shape such as a plate or film, or a flat porous membrane.

Hereinafter, the present invention will be described in detail with reference to embodiments for carrying out the present invention. Further, it is possible to implement the present invention by appropriately changing it within the scope of the gist of the present invention. Commercially available reagents were used unless otherwise specified.
<Polymer composition>
Proton nuclear magnetic resonance spectroscopy ( ¹ H-NMR) spectrum analysis using a nuclear magnetic resonance spectrometer (manufactured by JEOL Ltd., trade name JNM-GSX400), or nuclear magnetic resonance spectroscopy (manufactured by Bruker, trade name AVANCEIIIHD500). It was obtained from carbon nuclear magnetic resonance spectroscopy ( ¹³ C-NMR) spectroscopy using .
<Molecular weight and molecular weight distribution of the polymer>
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. Measured using 1,1,1,3,3,3-hexafluoro-2-isopropanol or N,N-dimethylformamide containing 10 mM lithium bromide. A measurement sample was prepared and measured at 1.0 mg/mL. Polymethyl methacrylate (manufactured by Polymer Laboratories) with a known molecular weight was used for the molecular weight calibration curve.

[Example 1]
As a substrate, a polystyrene dish (manufactured by Corning Incorporated) on which a layer of a temperature-responsive polymer was formed was used, and human iPS cells 201B7 strain were seeded at a density of 1300 cells/cm ² and incubated at 37°C at a CO ₂ concentration of 5. % environment. StemFit AK02N (manufactured by Ajinomoto Co., Inc.) was used as the medium. In addition, Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) and iMatrix-511 solution (manufactured by Nippi Co., Ltd.) were added to the medium until 24 hours after seeding the cells. 5 μL/mL) was added and cultured.

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After medium exchange 144 hours after seeding the cells, it was confirmed that the cells were present in the substrate in the form of colonies.

After removing the medium and washing with phosphate-buffered saline, cells were added with 0.5×TrypLE SELECT and ethylenediaminetetraacetic acid to phosphate-buffered saline at a concentration of 5×10 ⁻³ mol/L. The interjunction dissociation solution was added to the dish and treated at 37°C for 3 minutes. The intercellular junction dissociation solution was removed by aspiration, phosphate buffered saline was added, and the phosphate buffered saline was aspirated off to wash while the cells were adhered to the substrate, and then Y-27632 was added. The culture medium was added into the dish. Cells were detached by cooling at 15°C for 10 minutes. Cell viability was 96.5%.

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 degassing by nitrogen bubbling for 30 minutes, 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 to concentrate the reaction solution. The concentrate was poured into 250 mL of methanol, and the precipitated yellow oily substance was collected and dried under reduced pressure to obtain an n-butyl methacrylate polymer.

In a test tube, 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. Dissolved in 15 mL of dioxane. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 65° C. for 17 hours. After completion of the reaction, the reaction solvent was diluted with acetone and poured into 500 mL of hexane, and the precipitated pale yellow solid was collected 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 on a polystyrene dish at 2000 rpm to coat the temperature-responsive polymer.

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

[Example 2]
Using the base material prepared by the following method in Example 1, after cooling at 15 ° C. for 10 minutes, the base material was hit 20 times to apply vibration, and other steps were performed in the same manner as in Example 1 to exfoliate the cells. 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 the temperature-responsive polymer. The film thickness of the thermoresponsive polymer coating on the polystyrene dish was 5 nm.

[Comparative Example 1]
The procedure of Example 1 was repeated except that the polystyrene dish was used as it was without forming a layer of the temperature-responsive polymer. Cells did not detach from the substrate and could not be recovered.

[Comparative Example 2]
Cells were exfoliated in the same manner as in Example 1 except that the base material prepared by the following method was used in Example 1. Almost all of the cells were detached from the substrate during the contact between the pluripotent stem cells and the intercellular junction dissociation fluid, and the survival rate was 0%.

A 2 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 it with a temperature-responsive polymer. The film thickness of the thermoresponsive polymer coating on the polystyrene dish was 120 nm.

[Comparative Example 3]
The procedure of Example 1 was carried out in the same manner as in Example 1 except that the operation of contacting the pluripotent stem cells and the intercellular junction dissociation fluid was omitted. Only a part of the cells were detached from the substrate, and the detached cells were clusters of multiple cells, and single cells could not be recovered.

[Comparative Example 4]
In Example 1, the cells were detached from the substrate by performing a cooling detachment operation while trypsin remained without aspirating away the intercellular junction dissociation fluid that had been brought into contact with the pluripotent stem cells. Although the cells detached, most of the cells were dead with a viability of 29.5%.

Claims (3)

A method for exfoliating pluripotent stem cells cultured on a base material, wherein the base material has a layer of a temperature-responsive polymer with a product of layer thickness x temperature-responsive polymer component content ratio of 5 nm or more and 20 nm or less. A method for exfoliating pluripotent stem cells, characterized by comprising the following steps [1] to [3].
[1] A step of contacting an intercellular junction dissociation solution containing trypsin with pluripotent stem cells cultured on a substrate.
[2] A step of removing the intercellular junction dissociation solution containing trypsin while the pluripotent stem cells remain adhered to the substrate.
[3] A step of cooling the substrate and exfoliating the pluripotent stem cells from the substrate. The multipotent cell according to claim 1, wherein the contact time between the intercellular junction dissociation solution containing trypsin and the pluripotent stem cells adhered to the substrate in the step [1] is 1 second to 5 minutes. A method for exfoliating potential stem cells. 3. The method for exfoliating pluripotent stem cells according to claim 1, wherein the pluripotent stem cells are induced pluripotent stem cells.