Temperature-responsive substrate for cell culture and method for producing the same

Abstract

To provide a novel temperature-responsive surface.SOLUTION: A temperature-responsive substrate having a structure in which a water-insoluble polymer segment with the polymerization degree of 90 or more and a temperature-responsive polymer segment with the polymerization degree of 320 or more are bonded. A block polymer in which the content of the temperature-responsive polymer component is 50 to 95 wt.% is coated on a substrate surface as a temperature-responsive polymer portion in the ratio of 0.8 to 4.0 μg / cm2.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a cell culture substrate useful in fields such as biology and medicine. [Background technology]

[0002] Animal cell culture technology, including that of human cells, has advanced remarkably in line with the developments in various fields such as biology, medicine, drug discovery, and regenerative medicine. Research and development targeting these cells has also expanded to a wide range of fields. The uses of the animal cells have also evolved; initially, the cells themselves were commercialized as products, or their products were commercialized. Now, useful pharmaceuticals are being designed by analyzing the cells and their surface proteins, or the patient's own cells are being grown in vitro, or their function is being enhanced and they are being returned to the body for treatment. Currently, the technology of culturing animal cells is a field that is attracting the attention of many researchers.

[0003] Incidentally, most animal cells, including human cells, are adhesion-dependent. That is, when culturing animal cells outside of a living organism, it is necessary to first attach them to something. Against this backdrop, many researchers have previously designed and devised equipment surfaces that are more favorable to cells, but all of these technologies have been related to cell culture. When adhesion-dependent cultured cells attach to something, they produce adhesion proteins themselves. Therefore, when detaching these cells, conventional techniques require destroying these adhesion proteins, and this is usually done by enzymatic treatment. However, this has the significant problem that various cell-specific cell surface proteins produced by the cells during culture are also destroyed at the same time, yet in reality, there has been no way to solve this problem, and it has not been particularly considered. Solving this problem during cell retrieval is considered to be strongly required for the dramatic advancement of research and development targeting animal cells in the future.

[0004] Against this backdrop, Patent Document 1 describes a novel cell culture method in which cells are cultured on a cell culture support coated with a polymer having an upper or lower critical dissolution temperature of 0-80°C in water, at a temperature below the upper critical dissolution temperature or above the lower critical dissolution temperature, and then detached without enzymatic treatment by bringing the temperature above the upper critical dissolution temperature or below the lower critical dissolution temperature. Furthermore, Patent Document 2 describes using this temperature-responsive cell culture equipment to culture skin cells at a temperature below the upper critical dissolution temperature or above the lower critical dissolution temperature, and then detaching cultured skin cells with minimal damage by bringing the temperature above the upper critical dissolution temperature or below the lower critical dissolution temperature. In addition, Patent Document 3 describes a method for repairing surface proteins of cultured cells using this temperature-responsive cell culture equipment. By utilizing temperature-responsive cell culture equipment, various novel developments have become possible compared to conventional culture techniques.

[0005] The use of temperature-responsive cell culture equipment has enabled various novel developments compared to conventional cell culture techniques. However, this technology involves coating the surface of chemically inert engineering plastics using high energy such as electron beams, which requires large-scale equipment such as electron beam irradiation devices, and consequently, the cost of the substrate is inevitably high.

[0006] To address these challenges, several technologies have been developed. Representative examples include methods for synthesizing polymers with specific molecular structures, as described in Non-Patent Documents 1 and 2, and coating them onto the surface of a substrate. However, none of these technologies have reached the level of being able to culture cells in the same way as conventional cell culture substrates, detach cells simply by changing the temperature, as is the case with the temperature-responsive cell culture substrates made using electron beams, or detach the cultured cells as a cell sheet once they reach a confluent state. Therefore, there has been a need to improve the level of these technologies.

[0007] Furthermore, while Patent Document 4 proposed a temperature-responsive substrate coated with a block polymer having a structure in which water-insoluble polymer segments and temperature-responsive polymer segments are bonded, this technology still does not reach the level of being able to culture cells in the same way as conventional cell culture substrates, detach cells simply by changing the temperature in the same way as the temperature-responsive substrate for cell culture made using electron beams, or detach the cultured cells as a cell sheet when they reach a confluent state. Therefore, there was a need to improve this level of technology.

[0008] Similarly, Patent Document 5 also proposed a temperature-responsive substrate coated with a temperature-responsive polymer having a specific structure. However, this technology does not yet reach the level of being able to culture cells in the same way as conventional cell culture substrates, detach cells simply by changing the temperature in the same way as the temperature-responsive substrate for cell culture made using electron beams, or detach the cultured cells as a cell sheet when they reach a confluent state. Therefore, there was a need to improve this level of technology. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Application Publication No. 2-211865 [Patent Document 2] Japanese Patent Application Publication No. 05-192138 [Patent Document 3] Patent Application No. 2007-105311 [Patent Document 4] Patent No. 5846584 [Patent Document 5] Patent No. 6447787 [Non-patent literature]

[0010] [Non-Patent Document 1] Soft Matter, 5, 2937-2946 (2009) [Non-Patent Document 2] Interface, 4, 1151-1157 (2007) Summary of the Invention Problems to be Solved by the Invention

[0011] The present invention has been made with the intention of solving the problems of the prior art as described above. That is, an object of the present invention is to provide a novel temperature-responsive surface conceived from an idea completely different from that of the prior art. Another object of the present invention is to provide a method for using the same. Means for Solving the Problems

[0012] In order to solve the above problems, the present inventors conducted research and development by conducting studies from various angles. As a result, a block polymer having a structure in which a water-insoluble polymer segment with a degree of polymerization of 90 or more and a temperature-responsive polymer segment with a degree of polymerization of 320 or more are bonded, and having a temperature-responsive polymer component content of 50 to 95 wt%, is applied to the surface of a base material at a ratio of 0.8 to 4.0 μg / cm² in terms of the temperature-responsive polymer component to form a temperature-responsive base material for cell culture. It was found that cells to be cultured can well adhere to the surface of the base material, cells can be efficiently cultured, and moreover, the cells can be efficiently detached only by changing the temperature of the base material surface. The present inventors refer to the cultured cell sheet obtained by this method as an iMP cell sheet. The present inventors focused on the stereoregularity of a block copolymer having a structure in which a water-insoluble polymer and a temperature-responsive polymer are bonded, as well as the morphology and uniformity thereof on the surface of a base material, and greatly expect that using this as a base material for cell culture can provide a product with higher performance than the prior art temperature-responsive base material for cell culture. The present invention has been completed based on such findings.

[0013] That is, the present invention provides a temperature-responsive base material for cell culture, wherein a block polymer having a structure in which a water-insoluble polymer segment with a degree of polymerization of 90 or more and a temperature-responsive polymer segment with a degree of polymerization of 320 or more are bonded, and having a temperature-responsive polymer component content of 50 to 95 wt%, is coated on the surface of a base material at a ratio of 0.8 to 4.0 μg / cm² in terms of the temperature-responsive polymer component. The temperature-responsive substrate for cell culture according to the present invention is not limited, but preferred embodiments include the following.

[0014] In one aspect of the present invention, the temperature-responsive substrate for cell culture has a structure in which a water-insoluble polymer segment with a degree of polymerization of 90 or higher is bonded to a temperature-responsive polymer segment with a degree of polymerization of 320 or higher, and a block polymer having a temperature-responsive polymer component content of 50 to 95 wt% is coated on the substrate surface at a rate of 0.8 to 4.0 μg / cm2 as the temperature-responsive polymer component. In one aspect of the present invention, the temperature-responsive substrate for cell culture of the present invention is characterized in that the temperature-responsive polymer component of the block polymer is coated on the substrate surface within a predetermined amount of ±20%. In one aspect of the present invention, the temperature-responsive substrate for cell culture of the present invention is such that the monomer forming the water-insoluble polymer segment is butyl methacrylate or styrene. Furthermore, in one aspect of the present invention, the temperature-responsive substrate for cell culture of the present invention is characterized in that the monomer constituting the temperature-responsive polymer segment is N-isopropylacrylamide or contains the same. Furthermore, in one aspect of the present invention, the temperature-responsive substrate for cell culture of the present invention is characterized in that reversible addition-cleavage chain transfer agent sections from the production of the block polymer remain at the ends of the temperature-responsive polymer segments.

[0015] Furthermore, the present invention provides a method for producing a temperature-responsive substrate for cell culture. The method for producing the temperature-responsive substrate for cell culture of the present invention is not limited, but preferred embodiments include the following. In one aspect of the present invention, a method for producing a temperature-responsive substrate for cell culture is characterized by dissolving a block polymer in which a water-insoluble polymer segment with a degree of polymerization of 90 or higher is bonded to a temperature-responsive polymer segment with a degree of polymerization of 320 or higher, and in which the temperature-responsive polymer component content is 50 to 95 wt%, in an organic solvent, dropping the block polymer onto the substrate surface, and then uniformly coating and drying it by spin coating. In one aspect of the present invention, a method for producing a temperature-responsive substrate for cell culture is characterized in that the organic solvent is a mixture of isopropyl alcohol and N,N-dimethylformamide. In one aspect of the present invention, a method for producing a temperature-responsive substrate for cell culture is characterized in that the mixing ratio of isopropyl alcohol and N,N-dimethylformamide is 5:1 by volume. In one aspect of the present invention, a method for producing a temperature-responsive substrate for cell culture is characterized in that the block polymer is obtained by reversible addition-cleavage-chain transfer (RAFT) polymerization. In one aspect of the present invention, a method for producing a temperature-responsive substrate for cell culture is characterized in that the concentration of the block polymer solution dissolved in the organic solvent used for spin coating is 0.10% by mass to 0.30% by mass. In one aspect of the present invention, the method for producing a temperature-responsive substrate for cell culture involves dropping a block polymer solution dissolved in an organic solvent used for spin coating at a rate of 2.5 μl / cm² per unit area of ​​the substrate surface. 2 ~6.0 μl / cm³ 2 It is characterized by being dispensed by dripping. In one aspect of the present invention, a method for producing a temperature-responsive substrate for cell culture is characterized in that the spin coating begins to rotate 0 to 2 seconds after the block polymer solution is dropped onto the substrate surface. In one aspect of the present invention, a method for producing a temperature-responsive substrate for cell culture is characterized in that the spin coating rotation speed is 2500 rpm to 4500 rpm. [Effects of the Invention]

[0016] The substrate described in the present invention allows for good adhesion of cells to be cultured, enabling efficient cell culture. Furthermore, it allows for more efficient detachment compared to conventional techniques simply by changing the temperature of the substrate surface. In addition, substrates having such functional surfaces can be easily manufactured using the manufacturing method of the present invention. [Brief explanation of the drawing]

[0017] [Figure 1] This diagram shows the synthesis pathways for each product manufactured in Example 1. [Figure 2] This figure shows the results of culturing cells on the temperature-responsive surface obtained in Example 2. [Figure 3] This figure shows the results of culturing cells on the temperature-responsive surface obtained in Example 2. [Figure 4] This figure shows the results of culturing cells on the temperature-responsive surface obtained in Example 3. [Figure 5] This figure shows the results of culturing cells on the temperature-responsive surface obtained in Example 4. [Figure 6] This figure shows the results of culturing cells on the temperature-responsive surface obtained in Example 5. [Modes for carrying out the invention]

[0018] This invention relates to a block copolymer having a structure in which a water-insoluble polymer and a temperature-responsive polymer are bonded together, and the temperature-responsive polymer content is applied to the substrate surface at a concentration of 0.8 to 4.0 μg / cm³. 2 This is a temperature-responsive substrate for cell culture coated with a certain ratio. The water-insoluble polymer portion of the block polymer coated on the substrate surface has the effect of not being released from the substrate surface during cell culture, and also when detaching cultured cells by changing the temperature, which is a feature of the present invention. Furthermore, the temperature-responsive polymer portion has the effect of detaching cultured cells simply by cooling the culture medium from the temperature during culture (37°C) to 31°C or lower at the end of the culture period. At that time, the substrate surface contains 0.8 μg / cm³ of temperature-responsive polymer. 2If the concentration is lower, the cells are difficult to detach simply by cooling them after culture; conversely, 4.0 μg / cm³ is preferable. 2 If the amount is too high, the cells to be cultured cannot be properly attached, making it undesirable as a temperature-responsive substrate for cell culture according to the present invention. Furthermore, the more uniform the coating of the block polymer, the better the cells to be cultured can be attached, the more uniformly and efficiently the cells can be cultured on the substrate surface, and the more sensitively the cells can be detached simply by cooling, making it preferable as a temperature-responsive substrate for cell culture according to the present invention. To obtain such a substrate surface, it is preferable that the temperature-responsive polymer portion of the block polymer is coated on the substrate surface within a predetermined range of ±20%, preferably within a range of ±15%, even better within a range of ±10%, and most preferably within a range of ±5%. If the temperature-responsive polymer portion of the block polymer exceeds the predetermined range of ±20% on the substrate surface, the cells cannot be cultured uniformly on the substrate surface, and there will be areas where the cultured cells cannot be detached even when the culture medium is cooled, making it impossible to efficiently detach the cultured cells.

[0019] Here, the water-insoluble polymer should have a degree of polymerization of 90 or higher, preferably 95 or higher, and more preferably 100 or higher. While there is no particular upper limit to the degree of polymerization, it should be less than 130, preferably 120 or lower, and most preferably 110 or lower. If the degree of polymerization of the water-insoluble polymer is less than 90, a large amount of block polymer coated on the substrate surface will be released, which is undesirable for the temperature-responsive substrate for cell culture according to the present invention. In the present invention, after use in cell culture, the release rate of block polymer coated on the substrate surface is 10% or less as the temperature-responsive polymer portion of the block polymer, and the amount of block polymer released into the recovered cell suspension or cell sheet is extremely small, or undetectable by any analytical means. Conversely, it has been found that if the degree of polymerization of the water-insoluble polymer is greater than 130, the adhesion of the water-insoluble polymer to the substrate surface interferes, preventing the block polymer from spreading uniformly across the entire substrate surface. In other words, the water-insoluble polymer of the present invention has an excellent anchoring effect that prevents the release of block polymer during culture. Examples of such water-insoluble polymers in this invention include poly-n-butyl acrylate, poly-n-butyl methacrylate, poly-t-butyl methacrylate, and polystyrene.

[0020] On the other hand, the temperature-responsive polymer used in the present invention is preferably one with a degree of polymerization of 320 or higher, more preferably 330 or higher, more preferably 340 or higher, and most preferably 350 or higher. There is no particular upper limit to the degree of polymerization, but it is preferably less than 400, more preferably 380 or lower, more preferably 370 or lower, and most preferably 360 or lower. If the degree of polymerization of the water-insoluble polymer is less than 320, cells cultured on the substrate surface cannot be efficiently peeled off even when cooled, making it undesirable as a temperature-responsive substrate for cell culture of the present invention. Conversely, if the degree of polymerization of the temperature-responsive polymer exceeds 390, the viscosity of the polymer reaction solution increases drastically when producing the block polymer described below, making it impossible to sufficiently stir the reaction solution. As a result, the molecular weight distribution of the produced block polymer broadens, and even if it is coated on the substrate surface, it cannot adequately perform as a temperature-responsive substrate for cell culture of the present invention, which is undesirable. In the present invention, the content of the temperature-responsive polymer component in the temperature-responsive block polymer is preferably 50 to 95 wt%, more preferably 55 to 90 wt%, more preferably 60 to 85 wt%, and most preferably 65 to 80 wt%. If it is less than 50 wt%, the cultured cells on the polymer are difficult to detach even when the temperature is changed, which significantly reduces work efficiency and is undesirable. Conversely, if it is more than 95 wt%, cells are difficult to adhere to that area, making it difficult to sufficiently adhere cells, which is undesirable as a cell culture substrate for the present invention. Furthermore, the water-insoluble polymer portion of the block copolymer decreases, making it easier to detach from the substrate surface, which is undesirable. In the present invention, such a temperature-responsive polymer may be a polymer having a lower critical dissolution temperature (LCST), a homopolymer thereof, a copolymer, or a mixture thereof. Such a polymer can be obtained, for example, by the homopolymerization or copolymerization of the following monomers. Examples of monomers that can be used include (meth)acrylamide compounds and N-(or N,N-di)alkyl-substituted (meth)acrylamide derivatives, and in the case of copolymers, any two or more of these can be used.Furthermore, copolymerization with monomers other than those mentioned above, grafting or copolymerization of polymers, or mixtures of polymers and copolymers may be used. Crosslinking is also possible within a range that does not impair the inherent properties of the polymer. In this case, since the substance to be separated is a biomolecule, the separation is carried out in the range of 5°C to 50°C, so examples of temperature-responsive polymers include poly-Nn-propylacrylamide (lower limit critical dissolution temperature of the homopolymer is 21°C), poly-Nn-propylmethacrylamide (same 27°C), poly-N-isopropylacrylamide (same 32°C), poly-N-isopropylmethacrylamide (same 43°C), etc. The temperature-responsive polymer of the present invention preferably has a molecular weight of 3000 or more, preferably 5000 or more, more preferably 10000 or more, and most preferably 12000 or more. If the molecular weight is less than 3000, the cultured cells on the polymer are difficult to detach even when the temperature is changed, which is undesirable as it significantly reduces work efficiency.

[0021] The block copolymer of the present invention is a polymer formed by bonding a water-insoluble polymer with a water-affinity, temperature-responsive polymer. Therefore, when this block copolymer is coated onto a substrate surface and dried, it is expected to form a fine phase-separated structure on the substrate surface. The form and size of the phase-separated structure are not particularly limited, but it is advantageous that the presence of a phase-separated structure on the substrate surface can suppress cell degeneration when cells adhere to the substrate surface.

[0022] Furthermore, in the present invention, dithioester functional groups, which are part of the structure of the RAFT agent, remain at the ends of the generated polymer. This is a phenomenon characteristic of the RAFT polymerization method, and it is possible to start another polymerization reaction from the ends after the polymerization reaction is completed. In this case, the dithioester functional groups present at the ends of the temperature-responsive polymer can be easily replaced with thiol groups by adding 2-ethanolamine or the like. This reaction does not need to be carried out under special conditions, is simple, and proceeds in a short time. As a result, a polymer chain having highly reactive thiol groups can be obtained, and functional molecules having functional groups such as maleimide groups and thiol groups can be selectively and efficiently modified at the ends of the polymer chain. Therefore, it is possible to impart new functionality to the surface of the temperature-responsive culture substrate of the present invention. In this case, the type of functional group is not particularly limited, but examples include hydroxyl groups, carboxyl groups, amino groups, carbonyl groups, aldehyde groups, sulfonic acid groups, etc. Also, peptides or proteins that promote cell adhesion may be immobilized at the ends of the polymer chain. Furthermore, since the lower critical dissolution temperature (LCST) of poly-N-isopropylacrylamide changes depending on the hydrophilicity / hydrophobicity of the terminal functional groups, the introduction of functional groups to the polymer chain ends as in the present invention is also expected to be a new method for controlling the temperature responsiveness of the substrate surface from a different perspective.

[0023] The block copolymer of the present invention is obtained by growing a temperature-responsive polymer by radical polymerization in the presence of a RAFT agent using a reversible addition-cleavage chain transfer type radical polymerization (RAFT polymerization) method. The initiator used in this process is not particularly limited, but examples include 2,2'-azobis(isobutyronitrile), 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70), and 2,2'-azobis[(2-carboxyethyl)-2-(methylpropionamidine) (V-057)]. In the present invention, polymer chains are grown from this initiator. The RAFT agents used in this process are not limited to any particular type, but examples include benzyl dithiobenzoate, cumyl dithiobenzoate, 2-cyanopropyl dithiobenzoate, 1-phenylethylphenyl dithioacetate, cumylphenyl dithioacetate, benzyl 1-pyrrole carbodithioate, and cumyl 1-pyrrole carbodithioate.

[0024] The solvent used during polymerization in this invention is not particularly limited, but benzene, 1,4-dioxane, dimethylformaldehyde (DMF), etc., are preferred. This solvent is also not limited in any way, but can be appropriately selected depending on the type of monomer, RAFT agent, and polymerization initiator used in the polymerization reaction.

[0025] The present invention relates to a method for growing polymers with varying hydration strengths within a temperature range of 0 to 80°C by surface-initiated radical polymerization using a solvent such as 1,4-dioxane as an initiator in the presence of a RAFT agent. However, the initiator concentration, RAFT agent concentration, reaction temperature, reaction time, etc., during polymerization are not particularly limited and may be changed according to the purpose. Furthermore, the reaction solution may be left standing or stirred. The RAFT method of the present invention allows for easy purification of the product because it does not use metal ions, and the reaction process itself is convenient as it involves simple operations.

[0026] In this invention, the block copolymer obtained in this way is dissolved in a solvent and the copolymer is uniformly coated onto the surface of the substrate. The solvent used is not particularly limited as long as it dissolves the block copolymer, and examples include a mixed solvent of N,N-dimethylacrylamide and isopropyl alcohol, or a mixed solvent of acetonitrile and N,N-dimethylformamide. When multiple solvents are used in combination, the mixing ratio is not particularly limited, for example, in the case of a mixture of N,N-dimethylformamide and isopropyl alcohol, a ratio of 1 part N,N-dimethylformamide to 5 parts isopropyl alcohol and a ratio of 1 part N,N-dimethylformamide to 5 parts acetonitrile is preferable.

[0027] In the present invention, it is necessary to uniformly coat the above-mentioned block copolymer solution onto the substrate surface. In the present invention, as described above, it is necessary to uniformly coat the temperature-responsive block polymer, and a method using a spin coater is preferred. After coating, the temperature-responsive substrate for cell culture according to the present invention can be obtained by removing the solvent. At that time, the method of removing the solvent is not particularly limited, but examples include slowly evaporating it over time at room temperature / air, slowly evaporating it over time at room temperature / solvent-saturated environment, evaporating it by heating, and evaporating it by reducing the pressure. However, the first two methods are preferable for producing a uniform surface on the final temperature-responsive substrate for cell culture.

[0028] The inventors diligently investigated how to uniformly coat a temperature-responsive block polymer onto a substrate surface. As a result, they found that using isopropyl alcohol with N,N-dimethylformamide, which dissolves the block polymer well, significantly improves the properties of the substrate surface. Although the detailed reasons for this have not been fully elucidated, it is believed that the viscosity of the solution when the block polymer is dissolved and the spreading of the block polymer chains in the solution are good, and that the spin coating conditions of the present invention allow for uniform spreading on the substrate surface. Furthermore, they have found that the most preferable method for dissolving the block polymer in an organic solvent is to first dissolve the block polymer in N,N-dimethylformamide alone, and then add isopropyl alcohol to the solution.

[0029] The spin coating in the present invention is performed by dropping a solution of a temperature-responsive block polymer dissolved in an organic solvent onto the surface of a substrate. The concentration of the temperature-responsive block polymer solution at that time is preferably in the range of 0.10 wt% to 0.40 wt% (hereinafter, this wt% may be indicated as w%), preferably 0.15 wt% to 0.37 wt%, more preferably 0.20 wt% to 0.35 wt%, and most preferably 0.24 wt% to 0.33 wt%. If the concentration of the block polymer solution is less than 0.10 wt%, the amount of the block polymer remaining on the substrate surface after the subsequent spin coating does not reach the predetermined amount, and as a result, sufficient temperature responsiveness cannot be obtained, which leads to an unfavorable result for the present invention. Conversely, if the solution concentration exceeds 0.40 wt%, cell adhesion and subsequent proliferation are deteriorated, resulting in an unfavorable result for the present invention. In addition, when the concentration of this temperature-responsive block polymer solution is expressed as the ratio of the weight of the temperature-responsive block polymer to the volume of the organic solvent dissolving it, the concentration of the temperature-responsive block polymer solution is in the range of 0.10 mass volume% to 0.30 mass volume% (hereinafter, this mass volume% may be indicated as w / v%), preferably 0.12 mass volume% to 0.28 mass volume%, more preferably 0.15 mass volume% to 0.27 mass volume%, and most preferably 0.20 mass volume% to 0.25 mass volume%. If the concentration of the block polymer solution is less than 0.10 mass volume%, the amount of the block polymer remaining on the substrate surface after the subsequent spin coating does not meet the predetermined amount, and as a result, sufficient temperature responsiveness cannot be obtained, leading to an unfavorable result for the present invention. Conversely, if the solution concentration exceeds 0.3 mass volume%, cell adhesion and subsequent proliferation are deteriorated, resulting in an unfavorable result for the present invention. The dropping amount of the block polymer solution onto the substrate surface during spin coating is 2.0 μl / cm per unit area of the substrate surface 2 ~6.0 μl / cm 2 is preferably dropped, more preferably 2.5 μl / cm 2 ~5.0 μl / cm 2 is preferred, still more preferably 3.0 μl / cm 2 ~4.5 μl / cm 2A good ratio is, most preferably 3.5 μl / cm³. 2 ~4.2 μl / cm³ 2 The drop volume of the block polymer solution is 2.0 μl / cm³. 2 If the amount is too small, the block polymer cannot be uniformly coated onto the substrate surface after the subsequent spin coating, resulting in insufficient temperature response and an undesirable outcome for the present invention. Conversely, if the dropping amount is 6.0 μl / cm³ 2If the amount is increased, the block polymer on the substrate surface cannot be uniformly coated, and furthermore, the adhesion and subsequent proliferation of cells will be poor, resulting in an undesirable outcome according to the present invention. Also, for spin coating, it is best to start the rotation 0 to 2 seconds after the block polymer solution is dropped onto the substrate surface, preferably 0.2 to 1.8 seconds, more preferably 0.3 to 1.5 seconds, and most preferably 0.5 to 1.0 seconds. If the spin coater spins before 0 seconds after dropping (i.e., during dropping), the spin coating of the block polymer will not be uniform, resulting in uneven cell adhesion and proliferation, which is undesirable according to the present invention. Conversely, if the spin starts more than 2 seconds after dropping, the spin coating of the block polymer will not be uniform, resulting in uneven cell adhesion and proliferation, which is undesirable according to the present invention. Furthermore, the rotation speed during spin coating is preferably 2500 rpm to 4500 rpm, more preferably 2600 rpm to 4400 rpm, even more preferably 2800 rpm to 4300 rpm, and most preferably 3000 rpm to 4200 rpm. If the rotation speed is less than 2500 rpm, the adhesion and subsequent proliferation of cells will be poor, resulting in a product undesirable according to the present invention. Conversely, if the rotation speed is more than 4500 rpm, the amount of block polymer remaining on the substrate surface will not meet the predetermined amount, resulting in insufficient temperature responsiveness and a product undesirable according to the present invention. The present invention describes a method for spin coating a predetermined temperature-responsive block polymer onto a substrate surface, and the spin coating can be performed at room temperature of 10°C to 30°C, and it is desirable to perform it in a highly clean environment. The manufacturer, model, etc. of the spin coater are not limited in any way.

[0030] The material of the cell culture substrate to be coated is not limited to materials commonly used in cell culture, such as glass, modified glass, polystyrene, and polymethyl methacrylate, but can also be any material that can be shaped, such as polymer compounds other than those mentioned above, ceramics, and metals. Its shape is not limited to cell culture dishes such as petri dishes, but may also be a multi-plate, cell insert, fiber, or (porous) particle. Furthermore, it may have the shape of a container commonly used for cell culture (such as a flask).

[0031] The cells used on the temperature-responsive substrate surface for cell culture obtained in this invention may be any animal cells, and their source and preparation method are not particularly limited. Examples of cells in this invention include animal, insect, plant, and bacterial cells. In particular, examples of animal cell sources include humans, monkeys, dogs, cats, rabbits, rats, nude mice, mice, guinea pigs, pigs, sheep, Chinese hamsters, cattle, marmosets, African green monkeys, etc., but are not particularly limited. Furthermore, the culture medium used in this invention is not particularly limited as long as it is a culture medium for animal cells, but examples include serum-free medium and serum-containing medium. Such mediums may also have differentiation-inducing substances such as retinoic acid and ascorbic acid added. The seeding density on the substrate surface can be determined according to conventional methods and is not particularly limited.

[0032] Furthermore, with the temperature-responsive cell culture substrate of the present invention, cultured cells can be detached without enzymatic treatment by setting the temperature of the culture substrate above the upper critical dissolution temperature or below the lower critical dissolution temperature of the coating polymer on the culture substrate. This can be done in the culture medium or in other isotonic solutions, and the method can be selected according to the purpose. To detach and collect cells more quickly and efficiently, methods such as lightly tapping or shaking the substrate, or stirring the culture medium with a pipette, may be used alone or in combination.

[0033] By utilizing the temperature-responsive cell culture substrate described in this invention, cells obtained from various tissues can be cultured efficiently. This culture method allows for efficient and damage-free detachment simply by changing the temperature. Conventionally, such operations required time and skill from the operator, but this invention eliminates that need, enabling large-scale processing of cells. This invention demonstrates that such a culture substrate surface is produced by coating it with a specific temperature-responsive block polymer produced by living radical polymerization. In particular, following a reversible addition-cleavage chain transfer type radical polymerization method allows for simple and precise design of the culture substrate surface, and by continuing the reaction at the molecular chain ends, functional groups can be easily introduced, which is extremely advantageous for designing and producing cell culture substrate surfaces. [Examples]

[0034] The present invention will be described in more detail below based on examples, but these examples are not intended to limit the present invention in any way. [Examples]

[0035] A temperature-responsive block copolymer consisting of the temperature-responsive polymer poly-N-isopropylacrylamide (PIPAAm) and the hydrophobic polymer poly-n-butyl methacrylate (PBMA) was prepared by the method shown in Figure 1. Specifically, the hydrophobic polymer PBMA (specifically polymer species B90 (90-mer) to B130 (130-mer) in Figure 2), synthesized by reversible addition-cleavage chain transfer radical (RAFT) polymerization, was used as a macro-RAFT agent, and IPAAm was polymerized to it to produce various target temperature-responsive block polymers (PBMA-b-PIPAAm, specifically polymer species IP320 (320-mer) to IP380 (380-mer) in Figure 2) (Figure 1). The obtained polymers were evaluated by nuclear magnetic resonance spectroscopy (1H-NMR) and gel permeation chromatography (GPC) to confirm that the target temperature-responsive block polymers were obtained. [Examples]

[0036] The various PBMA-b-PIPAAm block polymers obtained in Example 1 were dissolved in an isopropyl alcohol / N,N-dimethylformamide mixed solvent (5 / 1 in v / v) at the concentrations shown in Figure 2. The block polymer solution was dropped onto the surface of a polystyrene substrate (3.5 cmφ dish) for cell culture, and spin-coated 0.5 seconds after dropping (rotation speed 4000 rpm, spin-coating time 30 sec). The amount of polymer on the surface of the obtained substrate was determined by total internal reflection Fourier transform infrared spectroscopy (ATR / FT-IR). As a result, for almost all polymer types, the amount of PIPAAm component on the substrate surface when using a polymer solution concentration of 0.15 wt% was 1.5 μg / cm³. 2 Similarly, when using a polymer solution at a concentration of 0.20% by weight, the amount of PIPAAm on the substrate surface is 2.0 μg / cm³. 2 When using a polymer solution at a concentration of 0.25% by weight, the amount of PIPAAm on the substrate surface is 2.5 μg / cm³. 2 When using a polymer solution at a concentration of 0.30% by weight, the amount of PIPAAm on the substrate surface is 3.0 μg / cm³. 2 When using a polymer solution at a concentration of 0.35% by weight, the amount of PIPAAm on the substrate surface is 3.5 μg / cm³. 2 The results were as follows. Furthermore, the measurement error for each PIPAAm component was within ±5%. NIH3T3 mouse fibroblasts were placed on the resulting substrate in a 1.0 x 10⁶ arrangement. 5Cells were seeded and cultured at a cell / 3.5cmφ dish concentration (culture medium: DMEM (containing 10% FBS), culture period: 4 days), and the adhesion of cells to the polymer-coated substrate surface at 37°C and the desorption behavior of cells due to low-temperature treatment at 20°C were evaluated (Figure 2). As a result, when spin-coating the temperature-responsive block polymer of the present invention onto the substrate surface, good cell adhesion and proliferation were observed when the polymer solution concentration was 0.20% to 0.30% by weight. After cooling at 20°C for 15 minutes, the cultured cells spontaneously desorbed from the substrate surface, forming a sheet-like structure (the inventors refer to this as an iMP cell sheet). As an example, Figure 3 shows the appearance of cultured cells when using a substrate with B100-IP350 and 0.25% by weight conditions. From these results, it is considered that the PIPAAm chains introduced onto the substrate surface are hydrated by low-temperature treatment below LCST, changing the properties of the substrate surface and causing the adhered cells to spontaneously detach. We are confident that the substrate surface coated with PBMA-b-PIPAAm will efficiently enable cell culture and subsequent detachment by changing the temperature. [Examples]

[0037] The PBMA-b-PIPAAm block polymer (B100-IP350) obtained in Example 1 was dissolved in an isopropyl alcohol / N,N-dimethylformamide mixed solvent (5 / 1 in v / v) to a concentration of 0.20% by weight. The block polymer solution was dropped onto the surface of a polystyrene substrate (3.5 cmφ dish) for cell culture and spin-coated under the conditions shown in Figure 4 (rotation speed 4000 rpm, spin-coating time 30 sec). The amount of polymer on the surface of the obtained substrate was determined by total internal reflection Fourier transform infrared spectroscopy (ATR / FT-IR). As a result, the amount of PIPAAm component on the substrate surface was almost consistently 2.0 μg / cm³ for each sample. 2 The measurement error for the amount of PIPAAm component between each sample was within ±5%. 1.0 x 10⁶ NIH3T3 mouse fibroblasts were placed on the resulting substrate. 5Cells were seeded and cultured at a cell / 3.5cmφ dish concentration (culture medium: DMEM (containing 10% FBS), culture period: 4 days), and the adhesion of cells to the polymer-coated substrate surface at 37°C and the desorption behavior of cells after low-temperature treatment at 20°C were evaluated (Figure 4). As a result, when spin-coating the substrate surface with the temperature-responsive block polymer of the present invention, good cell adhesion and proliferation were observed when spin-coating was started 0 to 2 seconds after dropping the polymer solution. After cooling at 20°C for 15 minutes, the cultured cells spontaneously desorbed from the substrate surface, and the cultured cells formed a sheet-like structure (the inventors refer to this as an iMP cell sheet). [Examples]

[0038] The various PBMA-b-PIPAAm block polymers obtained in Example 1 were dissolved in an isopropyl alcohol / N,N-dimethylformamide mixed solvent (5 / 1 in v / v) at the concentrations shown in Figure 5. The block polymer solution was dropped onto the surface of a polystyrene substrate (3.5 cmφ dish) for cell culture, and spin-coated 0.5 seconds after dropping (rotation speed 3000 rpm, spin-coating time 30 sec). The amount of polymer on the surface of the obtained substrate was determined by total internal reflection Fourier transform infrared spectroscopy (ATR / FT-IR). As a result, for almost all polymer types, the amount of PIPAAm component on the substrate surface when using a polymer solution concentration of 0.10 mass volume % was 1.0 μg / cm³. 2 Similarly, when using a polymer solution concentration of 0.15% by mass, the amount of PIPAAm on the substrate surface is 1.5 μg / cm³. 2 When using a polymer solution at a concentration of 0.20% by mass, the amount of PIPAAm on the substrate surface is 2.0 μg / cm³. 2 When using a polymer solution at a mass volume of 0.2.5%, the amount of PIPAAm component on the substrate surface is 2.5 μg / cm³. 2 When using a polymer solution at a concentration of 0.30% by mass, the amount of PIPAAm on the substrate surface is 3.0 μg / cm³. 2 The results were as follows. Furthermore, the measurement error for each PIPAAm component was within ±10%. 1.0 x 10⁶ NIH3T3 mouse fibroblasts were placed on the obtained substrate. 5Cells were seeded and cultured at a cell / 3.5cmφ dish concentration (culture medium: DMEM (containing 10% FBS), culture period: 4 days), and the adhesion of cells to the polymer-coated substrate surface at 37°C and the desorption behavior of cells upon low-temperature treatment at 20°C were evaluated (Figure 5). As a result, when spin-coating the temperature-responsive block polymer of the present invention onto the substrate surface, good cell adhesion and proliferation were observed when the polymer solution concentration was set to 0.10% by mass to 0.30% by mass. After cooling at 20°C for 15 minutes, the cultured cells spontaneously desorbed from the substrate surface, forming a sheet-like structure (which the inventors call an iMP cell sheet). From these results, it is considered that the PIPAAm chains introduced onto the substrate surface were hydrated by low-temperature treatment below LCST, changing the properties of the substrate surface and causing the adhered cells to spontaneously detach. We are confident that a substrate surface coated with PBMA-b-PIPAAm efficiently enables cell culture and desorption by changing the temperature. [Examples]

[0039] The PBMA-b-PIPAAm block polymer (B100-IP350) obtained in Example 1 was dissolved in an isopropyl alcohol / N,N-dimethylformamide mixed solvent (5 / 1 in v / v) to a concentration of 0.25% by mass. The block polymer solution was dropped onto the surface of a polystyrene substrate (3.5 cmφ dish) for cell culture and spin-coated under the conditions shown in Figure 6 (rotation speed 3000 rpm, spin-coating time 30 sec). The amount of polymer on the surface of the obtained substrate was determined by total internal reflection Fourier transform infrared spectroscopy (ATR / FT-IR). As a result, the amount of PIPAAm component on the substrate surface was almost consistently 2.5 μg / cm³ for each sample. 2 The measurement error for the amount of PIPAAm component between each sample was within ±10%. 1.0 x 10⁶ NIH3T3 mouse fibroblasts were placed on the resulting substrate. 5Cells were seeded and cultured at a cell / 3.5cmφ dish concentration (culture medium: DMEM (containing 10% FBS), culture period: 4 days), and the adhesion of cells to the polymer-coated substrate surface at 37°C and the desorption behavior of cells after low-temperature treatment at 20°C were evaluated (Figure 6). As a result, when spin-coating the substrate surface with the temperature-responsive block polymer of the present invention, good cell adhesion and proliferation were observed when spin-coating was started 0 to 2 seconds after dropping the polymer solution. After cooling at 20°C for 15 minutes, the cultured cells spontaneously desorbed from the substrate surface, and the cultured cells formed a sheet-like structure (the inventors refer to this as an iMP cell sheet). [Comparative Example 1]

[0040] The PBMA-b-PIPAAm block polymer (B100-IP350) obtained in Example 1 was dissolved in an isopropyl alcohol / N,N-dimethylformamide mixed solvent (5 / 1 in v / v) to a concentration of 0.05% by mass. The block polymer solution was dropped onto the surface of a polystyrene substrate (3.5 cmφ dish) for cell culture and spin-coated under the conditions shown in Example 4 (rotation speed 3000 rpm, spin-coating time 30 sec). The amount of polymer on the surface of the obtained substrate was determined by total internal reflection Fourier transform infrared spectroscopy (ATR / FT-IR). As a result, the amount of PIPAAm component on the substrate surface was 0.5 μg / cm³. 2 The measurement error for the amount of PIPAAm was within ±10%. 1.0 x 10⁶ NIH3T3 mouse fibroblasts were placed on the resulting substrate. 5 Cells were seeded at a concentration of 3.5 cmφ dishes and cultured (culture medium: DMEM (containing 10% FBS), culture period: 4 days). The adhesion of cells to the polymer-coated substrate surface at 37°C and the desorption behavior of cells after low-temperature treatment at 20°C were evaluated. As a result, although the cells showed good adhesion and proliferation, after cooling at 20°C for 15 minutes, the cultured cells did not spontaneously desorb from the substrate surface, making it unsuitable as a novel temperature-responsive cell culture substrate. [Comparative Example 2]

[0041] The PBMA-b-PIPAAm block polymer (B100-IP350) obtained in Example 1 was dissolved in an isopropyl alcohol / N,N-dimethylformamide mixed solvent (5 / 1 in v / v) to a concentration of 0.35% by mass. The block polymer solution was dropped onto the surface of a polystyrene substrate (3.5 cmφ dish) for cell culture and spin-coated under the conditions shown in Example 4 (rotation speed 3000 rpm, spin-coating time 30 sec). The amount of polymer on the surface of the obtained substrate was determined by total internal reflection Fourier transform infrared spectroscopy (ATR / FT-IR). As a result, the amount of PIPAAm component on the substrate surface was 3.5 μg / cm³. 2 The measurement error for the amount of PIPAAm was within ±15%. 1.0 x 10⁶ NIH3T3 mouse fibroblasts were placed on the resulting substrate. 5 Cells were seeded and cultured at a cell / 3.5cmφ dish concentration (culture medium: DMEM (containing 10% FBS), culture period: 4 days). The adhesion of cells to the polymer-coated substrate surface at 37°C and the desorption behavior of cells after low-temperature treatment at 20°C were evaluated. As a result, the cells exhibited uneven adhesion, resulting in poor cell proliferation, and the substrate was not suitable as a novel temperature-responsive cell culture substrate. [Industrial applicability]

[0042] By using the temperature-responsive cell culture substrate described in this invention, cells obtained from various tissues can adhere well to the substrate surface and be cultured. This culture method allows for easy and damage-free detachment of cultured cells without the need for enzymatic treatment. Compared to conventional temperature-responsive cell culture substrates, the substrate of this invention dramatically improves cell adhesion, proliferation, and detachability by cooling during cell culture.

Claims

1. Water-insoluble polymer segments with a degree of polymerization of 90 or higher and less than 130, and temperature-sensitive polymer segments with a degree of polymerization of 320 or higher and less than 380. A block polymer having a structure in which temperature-responsive polymer segments are bonded, with a temperature-responsive polymer component content of 50-95 wt%, is applied to the substrate surface as a temperature-responsive polymer component at a density of 0.8-3.0 μg / cm³. 2 A temperature-responsive substrate for cell culture, coated in a certain proportion.

2. The temperature-responsive substrate for cell culture according to claim 1, wherein the temperature-responsive polymer component of the block polymer is coated on the substrate surface within a predetermined amount within ±10% of that amount.

3. The temperature-responsive substrate for cell culture according to any one of claims 1 or 2, wherein the monomer forming the water-insoluble polymer segment is butyl methacrylate or styrene.

4. The temperature-responsive substrate for cell culture according to any one of claims 1 to 3, wherein the monomer constituting the temperature-responsive polymer segment is N-isopropylacrylamide or a material comprising the same.

5. A temperature-responsive substrate for cell culture according to any one of claims 1 to 4, wherein reversible addition-cleavage chain transfer agent sections from the production of the block polymer remain at the end of the temperature-responsive polymer segment.

6. A temperature-responsive substrate for cell culture according to any one of claims 1 to 5, wherein, after use in cell culture, the release rate of the block polymer coated on the substrate surface is 10% or less as the temperature-responsive polymer content of the block polymer.

7. The temperature-responsive substrate for cell culture according to any one of claims 1 to 6, wherein the substrate is a plastic petri dish, multiplate, flask, or cell insert.

8. Water-insoluble polymer segments with a degree of polymerization of 90 or higher and less than 130, and temperature-sensitive polymer segments with a degree of polymerization of 320 or higher and less than 380. A block polymer having a structure in which temperature-responsive polymer segments are bonded, and having a temperature-responsive polymer component content of 50-95 wt%, is dissolved in an organic solvent, and the block polymer is applied to the substrate surface. The process involves dropping the material onto a surface and then spin-coating it to ensure uniform application and drying. A method for producing a temperature-responsive substrate for cell culture according to any one of the requests 1 to 7.

9. The organic solvent is a mixture of isopropyl alcohol and N,N-dimethylformamide. A method for producing a temperature-responsive substrate for cell culture as described in item 8.

10. The dissolution of the block polymer in the organic solvent was carried out using N,N-dimethylformamide alone. The method for producing a temperature-responsive substrate for cell culture according to claim 8, wherein isopropyl alcohol is then added to the solution.

11. The mixing ratio of isopropyl alcohol and N,N-dimethylformamide is by volume. A method for producing a temperature-responsive substrate for cell culture according to any one of claims 8 to 10, wherein the ratio is 5:

1.

12. The block polymer is obtained by reversible addition-cleavage-chain transfer (RAFT) polymerization. A method for producing a temperature-responsive substrate for cell culture according to any one of claims 8 to 11.

13. A method for producing a temperature-responsive substrate for cell culture according to any one of claims 8 to 12, wherein the concentration of the block polymer solution dissolved in the organic solvent used for spin coating is 0.10% by mass to 0.30% by mass.

14. The amount of block polymer solution dissolved in the organic solvent used in the spin coating process is 2.0 μl / cm² per unit area of ​​the substrate surface. 2 ~6.0 μl / cm 2 A method for producing a temperature-responsive substrate for cell culture according to any one of claims 8 to 13, wherein the substrate is dispensed by drop.

15. A method for producing a temperature-responsive substrate for cell culture according to any one of claims 8 to 14, wherein the spin coating begins to rotate 0 to 2 seconds after the block polymer solution is dropped onto the substrate surface.

16. A method for producing a temperature-responsive substrate for cell culture according to any one of claims 10 to 15, wherein the spin coating rotation speed is 2500 rpm to 4500 rpm.

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