Cell culture method using temperature-responsive nonwoven fabric

Abstract

To provide a highly efficient cell culture method using a temperature-responsive nonwoven fabric containing a temperature-responsive polymer. [Solution] A method for culturing cells using a temperature-responsive nonwoven fabric containing a temperature-responsive polymer having a lower critical temperature (LCST) in the range of 0°C to 50°C, (1) A step of culturing the cells by adhering them to a temperature-responsive nonwoven fabric in a culture medium at a temperature of LCST or higher, (2) A step of cooling the temperature-responsive nonwoven fabric in the culture medium to below LCST and stirring or pipetting, (3) A step of adding a new temperature-responsive nonwoven fabric to the culture medium, (4) A step of adding a new culture medium to the culture medium, (5) The above problem is solved by including a step of culturing the cells again by bringing the culture medium, after adding a new temperature-responsive nonwoven fabric and a new culture medium, to a temperature of LCST or higher.

Description

[Technical Field] 【0001】 This disclosure relates to a cell culture method using a temperature-responsive nonwoven fabric. [Background technology] 【0002】 In recent years, the market for biopharmaceuticals and regenerative medicine using cells as raw materials has grown remarkably, creating a demand for technologies to efficiently culture raw materials in large quantities. Cells are classified into suspension cells and adherent cells, but most useful cells belong to the latter group. When culturing adherent cells, a substrate to serve as a scaffold is necessary, and conventionally, plastic dishes and flasks have been used. However, as the demand for cells expands, attention is being drawn to culture methods using cell culture scaffold materials suitable for large-scale culture. 【0003】 Among scaffolding materials for large-scale cell culture, nonwoven fabrics made of fibers have a high specific surface area and can provide a wide adhesion area to adherent cells. Therefore, by using nonwoven fabrics as scaffolding materials, it is possible to culture large quantities of cells more efficiently and in a smaller space. Product development using cells cultured in large quantities using nonwoven fabrics as raw materials, and product development using cell secretions derived from cultured cells as raw materials are progressing, leading to advancements in basic research on cell culture, tissue engineering, regenerative medicine, treatment of intractable diseases, vaccine production, and the development of cultured meat production technologies. 【0004】 One possible method for cell culture using nonwoven fabric is to add cells and nonwoven fabric to a culture medium, culture the resulting suspension in a culture vessel, adhere the cells to the surface of the nonwoven fabric, and then continuously agitate the culture medium to promote cell growth on the nonwoven fabric surface. To culture large quantities of cells using nonwoven fabric, it is necessary to perform subculturing and expansion culture. Methods for this include culturing cells, detaching them from the nonwoven fabric using proteolytic enzymes such as trypsin or protease, collecting only the cell suspension, and adding this cell suspension to a culture vessel containing new culture medium and new nonwoven fabric for expansion culture (enzymatic method), or culturing the cells until they reach a predetermined number of cells (for example, until they reach confluence), and then adding new nonwoven fabric to expand the cell culture (hereinafter also called the Fibers to Fibers Transfer method: FtoF method). 【0005】 The former enzymatic method had drawbacks, including being complicated and time-consuming, and the risk of damaging cells during enzymatic treatment and recovery. Furthermore, the three-dimensional structure unique to nonwoven fabrics made it difficult to detach cells and other organisms adhering between the fibers, resulting in reduced detachment efficiency, lower culture efficiency, / or lower cell viability, and / or a complicated process. 【0006】 In the latter FtoF method, cells adhering to the nonwoven fabric surface migrate to the new nonwoven fabric, resulting in uneven cell adhesion and / or a decrease in cell proliferation rate. 【0007】 To address the above issues, research has been conducted on creating nonwoven fabrics or fibrous cell culture scaffolds containing temperature-responsive materials. 【0008】 Patent Document 1 discloses temperature-responsive polymer fibers and nonwoven fabrics produced by electrospinning using temperature-responsive polymers. 【0009】 Patent Document 2 discloses a temperature-responsive substrate having a fibrous structure and a temperature-responsive polymer compound. 【0010】 In addition, Patent Document 3 and Non-Patent Document 1 disclose an example of producing a temperature-responsive non-woven fabric for cell culture by grafting the raw material monomers of a temperature-responsive polymer onto the surface of a non-woven fabric and polymerizing them by UV irradiation cross-linking. 【Prior Art Documents】 【Patent Documents】 【0011】 【Patent Document 1】 Japanese Patent Application Laid-Open No. 2009-057522 【Patent Document 2】 Japanese Patent Application Laid-Open No. 2008-029226 【Patent Document 3】 WO2022 / 019450 【Non-Patent Documents】 【0012】 【Non-Patent Document 1】 Ko et al., J. Biomater. Sci. Polym.Ed., 2018; 29(7-9): 1026-1041. 【Summary of the Invention】 【Problems to be Solved by the Invention】 【0013】 As an efficient method for large-scale culture, a method of subculturing is considered in which cells are adhered to a temperature-responsive non-woven fabric, cultured until a predetermined number of cells is reached, then the cells are detached by a cooling treatment, and thereafter a new temperature-responsive non-woven fabric is added (hereinafter, also referred to as the cooling Fibers to Fibers Transfer method: cooling FtoF method). 【0014】 However, since the temperature-responsive polymer fibers and non-woven fabrics disclosed in Patent Document 1 are shown to dissolve when the polymer fibers are placed in a constant-temperature bath at 3°C, it is considered difficult to re-adhere cells to the temperature-responsive polymer fibers or non-woven fabrics that have once dissolved after a subculture operation involving cell detachment by cooling treatment. Also, although polymer fibers that do not dissolve even when placed in a constant-temperature bath at 3°C are disclosed, it has been confirmed that those polymer fibers do not exhibit temperature responsiveness. Further, when the above temperature-responsive polymer fibers or non-woven fabrics are used in the cooling FtoF method, it is conceivable that when the temperature is raised to the cell culture temperature (e.g., 37°C) after the cooling treatment, the temperature-responsive polymer fibers or non-woven fabrics that have once dissolved due to the cooling treatment will precipitate again. However, at that time, it is conceivable that they will aggregate or non-specifically adsorb to the surface of the newly added non-woven fabric due to hydrophobic interactions or the like. Then, it is considered that the cells will adhere not only to the newly added non-woven fabric but also to the aggregates and the precipitates adsorbed to the newly added non-woven fabric, and each cell will adhere to a different substance, so it is considered difficult to stably and uniformly produce cells. 【0015】 Also, since the structure of the temperature-responsive substrate disclosed in Patent Document 2 collapses by changing the temperature from a temperature higher than the lower critical solution temperature of the temperature-responsive polymer compound to a temperature lower than the lower critical solution temperature in an aqueous solution, the same problems as those in Patent Document 1 are suggested. 【0016】 Since the polymerization method of the temperature-responsive non-woven fabric for cell culture disclosed in Patent Document 3 and Non-Patent Document 1 is a method of imparting temperature-responsive characteristics only to a specific surface of the non-woven fabric, it is difficult to subculture the cells on the entire non-woven fabric by a cooling operation because cell detachment by cooling cannot be performed at locations other than the specific surface to which the temperature responsiveness of the non-woven fabric is imparted. That is, it has been suggested that the cooling FtoF method is difficult in a three-dimensional culture system in which cells are adhered to the entire non-woven fabric. 【0017】 The object of this disclosure is to provide a highly efficient cell culture method using a temperature-responsive nonwoven fabric containing a temperature-responsive polymer. One aspect of this disclosure is to provide a cell culture method using a temperature-responsive nonwoven fabric containing a temperature-responsive polymer, wherein the cell culture is performed by a cooled FtoF method. Another aspect of this disclosure is to provide a cell culture method using a temperature-responsive nonwoven fabric containing a temperature-responsive polymer, wherein the cell culture is performed by a cooled FtoF method in a three-dimensional culture system. [Means for solving the problem] 【0018】 As a result of diligent research, the present inventors have discovered that cells can be cultured with high efficiency in a cell culture method using a temperature-responsive nonwoven fabric containing a temperature-responsive polymer, by performing a subculturing operation that includes a cooling step and a stirring or pipetting step. In other words, this disclosure encompasses the following aspects. [1] A method for culturing cells using a temperature-responsive nonwoven fabric containing a temperature-responsive polymer having a lower critical temperature (LCST) in the range of 0°C to 50°C, A step of culturing cells by adhering them to a temperature-responsive nonwoven fabric in a culture medium at a temperature of LCST or higher, The steps of cooling the temperature-responsive nonwoven fabric in the culture medium to below LCST and stirring or pipetting, A step of adding a new temperature-responsive nonwoven fabric to the culture medium, A step of adding a new culture medium to the culture medium, and The process includes a step of culturing the cells again by bringing the culture medium, after adding a new temperature-responsive nonwoven fabric and a new culture medium, to a temperature above the LCST. A method wherein the temperature-responsive polymer is a block copolymer comprising the blocks (A), (B), and (C) below; (A) Polymer blocks with an HLB value (Griffin method) in the range of 7 to 20. (B) Polymer blocks with an HLB value (Griffin method) in the range of 0 or more and less than 7. (C) A temperature-responsive polymer block in which LCST is in the range of 0°C to 50°C. [2] A method for culturing cells using a temperature-responsive nonwoven fabric containing a temperature-responsive polymer having an LCST in the range of 0°C to 50°C, A step of culturing cells by adhering them to a temperature-responsive nonwoven fabric in a culture medium at a temperature of LCST or higher, The process involves adding a new culture medium below LCST to the culture medium, thereby cooling the temperature-responsive nonwoven fabric to below LCST, and then stirring or pipetting it. A step of adding a new temperature-responsive nonwoven fabric to the culture medium, The process includes a step of culturing the cells again by bringing the culture medium, after adding a new culture medium and a new temperature-responsive nonwoven fabric, to a temperature above LCST. A method wherein the temperature-responsive polymer is a block copolymer comprising the blocks (A), (B), and (C) below; (A) Polymer blocks with an HLB value (Griffin method) in the range of 7 to 20. (B) Polymer blocks with an HLB value (Griffin method) in the range of 0 or more and less than 7. (C) A temperature-responsive polymer block in which LCST is in the range of 0°C to 50°C. [3] The method according to either [1] or [2], wherein the temperature-responsive polymer comprises a block polymer of 2-methoxyethyl acrylate, n-butyl acrylate, and N-isopropylacrylamide. [4] A step of cooling the temperature-responsive nonwoven fabric in the culture medium to below LCST and stirring or pipetting, A step of adding a new temperature-responsive nonwoven fabric to the culture medium, A step of adding a new culture medium to the culture medium, and The method according to [1], wherein the step of culturing the cells again is repeated two or more times by bringing the culture medium, after adding a new temperature-responsive nonwoven fabric and a new culture medium, to a temperature of LCST or higher. [5] A step of adding a new culture medium below LCST to the culture medium to cool the temperature-responsive nonwoven fabric below LCST, and stirring or pipetting, A step of adding a new temperature-responsive nonwoven fabric to the culture medium, The method according to [2], wherein the step of culturing the cells again is repeated two or more times by bringing the culture medium, after adding a new culture medium and a new temperature-responsive nonwoven fabric, to a temperature of LCST or higher. [Effects of the Invention] 【0019】 According to this disclosure, in a cell culture method using a temperature-responsive nonwoven fabric containing a temperature-responsive polymer, cell culture can be achieved with high efficiency by performing a subculturing operation that includes a cooling step and a stirring step. Furthermore, according to this disclosure, since the temperature-responsive nonwoven fabric of this disclosure can maintain its shape even with temperature changes, cells can adhere again if the temperature is raised to above the lower critical temperature (LCST) even after the cells have detached by cooling to a temperature below the LCST. 【0020】 Furthermore, according to one aspect of this disclosure, a cell culture using a temperature-responsive nonwoven fabric containing a temperature-responsive polymer is possible, and the cell culture can be performed by the cooling FtoF method. 【0021】 Furthermore, according to one aspect of this disclosure, a cell culture using a temperature-responsive nonwoven fabric containing a temperature-responsive polymer is possible, which allows cell culture in a three-dimensional culture system by a cooled FtoF method. 【0022】 Therefore, this disclosure provides a method for efficiently culturing cells, which is useful for industrial cell culture. [Brief explanation of the drawing] 【0023】 [Figure 1] A diagram comparing the number of Vero cells based on differences in nonwoven fabric and subculturing methods. [Modes for carrying out the invention] 【0024】 The following describes in detail embodiments for carrying out the present invention, but this is not intended to limit the present invention to the following. The present invention can be implemented with appropriate modifications within the scope of its spirit. 【0025】 In this specification, nonwoven fabric refers to a fabric made by forming fibers into a cloth and bonding the fibers together. There are no particular limitations on the sheeting method for forming the nonwoven fabric, but generally examples include the dry method formed in air, the wet method formed in water, and the spunbond method. Similarly, there are no particular limitations on the bonding method for forming bonds between fibers, but generally examples include thermal bonding, chemical bonding, and mechanical bonding, which can be appropriately selected depending on ease of processing and application. 【0026】 The true density of the raw materials for the fibers that make up the nonwoven fabric is 1.01 to 2.00 g / cm³ for settling in water. 3 It may be such that, preferably 1.01 to 1.5 g / cm³, so that it disperses easily under stirring. 3 This may be, and more preferably, 1.01 to 1.2 g / cm³. 3 That's fine. 【0027】 The water absorption rate of the fibers constituting the nonwoven fabric is 0.2% or less, preferably 0.15% or less, and more preferably 0.1% or less, in order to maintain strength without decomposing in water. For the evaluation method of water absorption rate, refer to JIS K 7209:2000, Plastics - Determination of water absorption, which is a translation of ISO 62:1999, Plastics - Determination of water absorption. 【0028】 While there are no particular limitations on the material of the fibers constituting the nonwoven fabric, examples of materials within the range of true density of the raw materials and water absorption rate of the fibers constituting the nonwoven fabric of this disclosure include polycarbonate, polyethylene terephthalate, polybutylene terephthalate, polyvinyl chloride, polyetheretherketone, and polyphenylene sulfide. However, polyethylene terephthalate is preferred due to its ease of processing. Furthermore, as long as the material is within the above-mentioned range of true density and water absorption rate, it may be made of copolymers or multilayer yarns. 【0029】 There are no particular limitations on the thickness of the fibers that make up the nonwoven fabric, but preferably, in order for cells to adhere, The particle size is 1 to 1000 μm, and more preferably 10 to 300 μm. 【0030】 The structure of the fibers that make up the nonwoven fabric is not particularly limited, and one example of a cross-sectional structure is circular. To increase the surface area, you can use flower-shaped or flattened multi-leaf types, or ensure the growing medium is distributed throughout the entire nonwoven fabric. Therefore, a hollow fiber structure is also acceptable. 【0031】 There are no particular limitations on the mesh size of the nonwoven fabric; for example, it may be 1 μm to 1000 μm, and more preferably 1 to 300 μm. However, it is preferable to select the appropriate mesh size considering the cell type used, culture characteristics, and characteristics of the culture vessel. 【0032】 There are no specific limits on the specific surface area of ​​nonwoven fabrics, but as an example, it ranges from 100 to 10,000 cm². 2 It may be / g, preferably 300-8000 cm 2 / g, more preferably 500-5000cm 2 It may be / g. A smaller specific surface area of ​​the nonwoven fabric may reduce its strength and make it difficult to handle, while a larger specific surface area per unit area may decrease the porosity of the nonwoven fabric. Therefore, it is preferable to select the appropriate value considering the cell type used, culture characteristics, and characteristics of the cultured cells. 【0033】 A temperature-responsive polymer, which is one of the components of the temperature-responsive nonwoven fabric used in the method of the present disclosure, is a block copolymer comprising the blocks (A), (B), and (C) below; (A) Polymer blocks with an HLB value (Griffin method) in the range of 7 to 20. (B) Polymer blocks with an HLB value (Griffin method) in the range of 0 or more and less than 7. (C) A temperature-responsive polymer block in which LCST is in the range of 0°C to 50°C. 【0034】 In this specification, the Lower Critical Solution Temperature (LCST; hereinafter sometimes referred to as LCST) is the temperature at which, below which the polymer dissolves in water to form a clear solution, but above which it becomes insoluble, resulting in turbidity or precipitation and phase separation. The LCST is not particularly limited, but is preferably near the culture temperature, for example, in the range of 0°C to 50°C, preferably 20°C to 40°C, and more preferably in the range of 25°C to 35°C. 【0035】 Examples of homopolymers exhibiting LCST and their LCSTs in water include N-isopropylacrylamide (LCST=32°C), Nn-propylmethacrylamide (LCST=22°C), N-tetrahydrofurfurylacrylamide (LCST=28°C), N-ethoxyethylacrylamide (LCST=35°C), N,N-diethylacrylamide (LCST=32°C), Nn-propylmethacrylamide (LCST=28°C), N-tetrahydrofurfurylmethacrylamide (LCST=35°C), N-methyl-N-isopropylacrylamide (LCST=23°C), or N-methyl-Nn-propylacrylamide (LCST=20°C). The lower critical temperature varies depending on the concentration of the aqueous solution, but N-isopropylacrylamide is preferred because its lower critical temperature is less dependent on concentration. 【0036】 Block (C) may use only one type of repeating unit, or it may use two or more types in combination. Furthermore, if it is temperature responsive, it may contain different repeating units in addition to the temperature responsive repeating unit. Furthermore, if it has LCST, in addition to the repeating units of the polymer that exhibits LCST, it may also contain repeating units of the polymer that does not exhibit LCST, depending on the purpose, such as improving adhesion to cells or temperature responsive nonwoven fabrics. The composition of N-isopropylacrylamide exhibiting LCST among the polymers is preferably 10 mol% to 95 mol%, preferably 30 mol% to 80 mol%, and more preferably 65 mol% to 70 mol%. 【0037】 HLB (Hydrophile-Lipophile Balance: HLB) is a value representing the degree of affinity to water and oil, as described in WCGriffin, Journal of the Society of Cosmetic Chemists, 1, 311 (1949). It takes a value from 0 to 20, with values ​​closer to 0 indicating higher hydrophobicity and values ​​closer to 20 indicating higher hydrophilicity. There are several methods for calculating HLB using formulas, including the Atlas method, Griffin method, Davis method, and Kawakami method. In this specification, however, the value calculated by the Griffin method may be used, and the HLB may be calculated using the following formula based on the formula weight of the hydrophilic portion in the repeating unit of each block constituting the block copolymer and the total formula weight of the repeating unit. [Mathematics 1] HLB value = 20 × (formula weight of the hydrophilic part in the repeating unit) ÷ (total formula weight of the repeating unit) Examples of the hydrophilic portion in the repeating units of each block mentioned above include the sulfone portion (-SO3-), phosphono portion (-PO3-), carboxyl portion (-COOH), ester portion (-COO-), amide portion (-CONH-), imide portion (-CON-), aldehyde portion (-CHO), carbonyl portion (-CO-), hydroxyl portion (-OH), amino portion (-NH2), acetyl portion (-COCH3), ethyleneamine portion (-CH2CH2N-), ethyleneoxy portion (-CH2CH2O-), alkali metal ions, alkaline earth metal ions, ammonium ions, halide ions, and acetate ions. 【0038】 In calculating the hydrophilic portion within a repeating unit, atoms constituting the hydrophilic portion must not overlap with atoms constituting other hydrophilic portions. Examples of HLB value calculations within repeating units are given below. For example, in the case of 2-methacryloyloxyethyl phosphorylcholine (molecular weight: 295.27), the hydrophilic portion consists of 1 part ester, 1 part phosphono group, and 1 part ethyleneamine, and the molecular weight of the hydrophilic portion is 181.04, so the HLB value is 12.3. In the case of 2-dimethylaminoethyl methacrylate (molecular weight: 157.11), the hydrophilic portion consists of 1 part ester and 1 part ethyleneamine, and the molecular weight of the hydrophilic portion is 86.07, so the HLB value is 11.0. In the case of methyl methacrylate (molecular weight: 100.12), the hydrophilic portion consists of 1 part ester, and the molecular weight of the hydrophilic portion is 44.01, so the HLB value is 8.8. In the case of n-butyl methacrylate (molecular weight: 142.20), the hydrophilic portion consists of 1 part ester portion, and the molecular weight of the hydrophilic portion is 44.01, so the HLB value is 6.2. 【0039】 An example of a block copolymer containing blocks (A), (B), and (C) is a block copolymer consisting of repeating units of (A) 2-methoxyethyl acrylate (HLB value = 13.5), (B) n-butyl acrylate (HLB value = 6.9), and (C) N-isopropylacrylamide (LCST = 32°C). 【0040】 For the functional polymer to be fixed to the nonwoven fabric, it is preferable that the number-average molecular weight of the functional polymer be above a certain value, and it is also preferable that the number-average molecular weight of the functional polymer be below a certain value in order to lower the viscosity of the functional polymer solution. An example of the number-average molecular weight of the functional polymer is 1,000 to 1,000,000, preferably 5,000 to 500,000, more preferably 10,000 to 200,000, and particularly preferably 50,000 to 100,000. The number-average molecular weight of the functional polymer can be measured, for example, by gel permeation chromatography (GPC). Gel permeation chromatography (GPC) can be performed, for example, under the conditions described in the examples below. 【0041】 Furthermore, the temperature-responsive nonwoven fabric of this disclosure may contain other polymer compounds in addition to polymers exhibiting LCST, depending on the purpose, such as improving adhesion to cells or to the temperature-responsive nonwoven fabric. For example, to improve cell adhesion, it is preferable to include a polymer compound consisting of carboxystyrene and styrene. 【0042】 There are no particular limitations on the method for manufacturing a temperature-responsive nonwoven fabric, but a method that can introduce a temperature-responsive polymer throughout the nonwoven fabric is preferable because it can better utilize the properties of the nonwoven fabric, which has a high specific surface area, and thus enable more efficient cell culture. One example of a method that can introduce a temperature-responsive polymer throughout the nonwoven fabric is to apply a surface treatment agent, in which a temperature-responsive polymer is dissolved in a solvent, to the nonwoven fabric and physically coat it. Examples of methods for applying a temperature-responsive polymer to a nonwoven fabric include a method that includes an immersion treatment in which the nonwoven fabric is immersed in a temperature-responsive polymer solution for a certain period of time and the functional polymer is fixed to the nonwoven fabric by simply separating the solvent from the nonwoven fabric; a method that includes an impregnation treatment in which the nonwoven fabric is immersed in a functional polymer solution and the functional polymer is fixed to the nonwoven fabric by evaporating and removing the solvent component by heating, standing and stirring or more; and a method that includes a vacuum impregnation treatment in which the nonwoven fabric is immersed in a functional polymer solution and the solvent component is evaporated and removed by reducing the pressure. The temperature-responsive polymer may also be applied to the nonwoven fabric by combining the above methods. 【0043】 According to the method disclosed herein, a temperature-responsive nonwoven fabric can be used for cell culture. The method disclosed herein is particularly preferred for use with adherent cells. Adherent cells are cells that proliferate while adhering to the surface of a cell culture substrate such as a nonwoven fabric. The origin of the cells is not particularly limited, but examples include mammals such as humans, monkeys, dogs, cats, rabbits, rats, nude mice, mice, guinea pigs, pigs, sheep, Chinese hamsters, and cows, as well as birds such as chickens and ducks. Specific cell types include, for example, CHO cells derived from Chinese hamster ovaries, Vero cells derived from African green monkey kidneys, L929 cells derived from mouse connective tissue, MDCK cells derived from dog kidneys, HEK293 cells derived from human fetal kidneys, MRC-5 cells derived from normal human fetal lung tissue, CEF cells derived from chicken fetuses, BHK-21 cells derived from hamster kidneys, MDBK cells derived from bovine kidneys, CRFK cells derived from cat kidneys, CPK cells derived from guinea pig kidneys, and HeLa cells derived from human cervical cancer, among other cultured cell lines, as well as epithelial cells and endothelium that make up various tissues and organs in the body. Examples of cells include contractile skeletal muscle cells, smooth muscle cells, cardiomyocytes, neurons, glial cells, fibroblasts that constitute the nervous system, macrophages and dendritic cells involved in the body's immunity, hepatocytes, non-parenchymal hepatocytes and adipocytes involved in the body's metabolism, and cells with differentiation potential such as induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic carcinoma (EC) cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, germline stem cells, and other stem cells, as well as progenitor cells of various tissues, and cells differentiated from these. Mesenchymal stem cells are particularly preferred. Mesenchymal stem cells refer to a group of stem cells and their progenitor cells that can differentiate into all or some mesenchymal cells such as chondrocytes, osteoblasts, and adipocytes. There are no particular limitations on the origin of mesenchymal stem cells, but examples include those derived from tissues such as bone marrow, fat, dental pulp, umbilical cord blood, placenta, and synovial membrane, as well as pluripotent stem cells such as ES cells and iPS cells. In addition to the cells mentioned above, other examples include cells that have been appropriately genetically modified according to their intended purpose. 【0044】 The type of culture medium used in the culture method disclosed herein is not particularly limited. For example, basal media such as MEM, αMEM, DMEM, EMEM, GMEM, DMEM / Ham'sF-12, Ham'sF-12, Ham'sF-10, Medium199, and RPMI1640 may be used, as well as serum media (basal media with added serum) or serum-free media. The serum used is not particularly limited. For example, fetal bovine serum (FBS), calf serum, adult cow serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rabbit serum, and human serum may be used. The culture medium may or may not contain antibiotics. In addition, additives other than serum and antibiotics may be appropriately selected and added at appropriate concentrations depending on the cell type. 【0045】 The present disclosure is a method for culturing cells using a temperature-responsive nonwoven fabric containing a temperature-responsive polymer having a lower critical temperature (LCST) in the range of 0°C to 50°C, (1) A step of culturing the cells by adhering them to a temperature-responsive nonwoven fabric in a culture medium at a temperature of LCST or higher, (2) A step of cooling the temperature-responsive nonwoven fabric in the culture medium to below LCST and stirring or pipetting, (3) A step of adding a new temperature-responsive nonwoven fabric to the culture medium, (4) A step of adding a new culture medium to the culture medium, (5) A step of culturing the cells again by raising the culture medium, after adding a new temperature-responsive nonwoven fabric and a new culture medium, to a temperature of LCST or higher. Includes. 【0046】 Furthermore, the present disclosure relates to a method for culturing cells using a temperature-responsive nonwoven fabric containing a temperature-responsive polymer having an LCST in the range of 0°C to 50°C, (I) A step of culturing the cells by adhering them to a temperature-responsive nonwoven fabric in a culture medium at a temperature of LCST or higher. (II) A step of adding a new culture medium below LCST to the culture medium to cool the temperature-responsive nonwoven fabric below LCST, and stirring or pipetting it. (III) A step of adding a new temperature-responsive nonwoven fabric to the culture medium, (VI) A step of culturing the cells again by raising the culture medium, after adding a new culture medium and a new temperature-responsive nonwoven fabric, to a temperature of LCST or higher. Includes. 【0047】 The temperature used for cell culture in (1), (5), (I), and (VI) above is not particularly limited as long as it is above LCST. However, when using mammalian cells such as human or animal cells, it is acceptable to culture at around body temperature to obtain high culture efficiency. For example, a temperature range of 30°C to 40°C is preferred, and a temperature range of 36°C to 38°C is even more preferred. 【0048】 The method of culturing cells in (1), (5), (I), and (VI) above is not particularly limited and can be carried out by either leaving the nonwoven fabric to which the cells are attached still in the culture medium or by suspending it in the culture medium. Since good dispersion conditions for nutrients and cell waste in the culture medium are desirable, it is preferable to carry out the culture by suspending the nonwoven fabric. The method of culturing by suspending the nonwoven fabric can be carried out by, for example, a screw-type agitator, a vane-type agitator, shaking, up-and-down reciprocating agitator, or a combination of the above methods. When culturing under shaking conditions, there are no particular limitations on the shaking speed or the direction of shaking. Considering the usefulness on an industrial scale, stirring culture is more preferable. 【0049】 The stirring speed at which the nonwoven fabric can be suspended in the culture medium depends on the shape and capacity of the culture vessel, the stirring method, and the true density of the nonwoven fabric. As an example, in the case of a bioreactor with a 1-50 L impeller-type stirring tank, it is preferable to use a stirring speed of 1-500 rpm, 1-300 rpm, 1-200 rpm, 1-100 rpm, 1-90 rpm, 1-80 rpm, 1-70 rpm, 1-60 rpm, or 1-50 rpm. There are no particular limitations on the culture temperature during cultivation, but it is preferable to use a temperature of LCST or higher, which allows the surface of the temperature-responsive polymer-coated nonwoven fabric to become hydrophobic and provides an environment where cells can easily adhere. The concentration of the non-woven fabric in the culture solution can be appropriately adjusted based on the cultured cell type, cell seeding density, stirring speed, and the size and specific gravity of the non-woven fabric, etc. For example, it can be set to 0.01 - 100 g / L, 0.1 - 50 g / L, 0.5 - 20 g / L, or 1 - 10 g / L. 【0050】 The seeding density of the cells is not particularly limited as long as the cells adhere and proliferate. In the case of human-derived mesenchymal stem cells, for example, 1.0×10 1 cells / cm 2 ~1.0×10 5 cells / cm 2 is preferable, and 1.0×10 2 cells / cm 2 ~1.0×10 4 cells / cm 2 is more preferable. In the case of Vero cells, for example, 1.0×10 1 cells / cm 2 ~1.0×10 5 cells / cm 2 is preferable, and 5.0×10 2 cells / cm 2 ~5.0×10 3 ]>cells / cm 2 is more preferable. Other culture conditions are not particularly limited, and culturing may be carried out under conditions commonly used in the art. 【0051】 The cell culture container is not particularly limited, but a container that can be stirred and mixed is more preferable. The cell culture container may have an internal volume of 5000 L or less, 3000 L or less, 1000 L or less, 500 L or less, 300 L or less, 100 L or less, 50 L or less, 10 L or less, 5 L or less, 1 L or less, 500 mL or less, 100 mL or less, 50 mL or less, or 30 mL or less. 【0052】 The method for cooling the temperature-responsive nonwoven fabric to below LCST in (2) above is not particularly limited. It may be cooled directly using a cooler, or the temperature-responsive nonwoven fabric may be cooled by adding phosphate buffer or the like cooled to below LCST as a cooling solution to the culture medium, or, as described in (II), the temperature-responsive nonwoven fabric may be cooled by adding a new medium cooled to below LCST as a cooling solution to the culture medium. Adding cooled phosphate buffer to the culture medium is preferable because it makes it easier for cells to detach from the nonwoven fabric. Including a chelating agent such as EDTA or EGTA in the cooling solution is preferable because it can detach cell adhesion, making it easier to collect cells as single cells. Here, a single cell means that the cells have separated from each other and are no longer connected. The concentration of the chelating agent is not particularly limited and may be 1 μmol / L to 1 mmol / L. 【0053】 When cooling and detaching cells in (2) and (II) above, it is preferable to obtain fluidity in the culture medium by stirring or pipetting, as this can promote the cooling of the temperature-responsive nonwoven fabric. In other words, stirring or pipetting should be included in the cooling process to improve cell detachment. There are no particular limitations on the stirring method, but examples include screw-type stirring, vane-type stirring, shaking stirring, and up-and-down reciprocating stirring. It is preferable to select an appropriate method considering the cell type used, culture characteristics, and characteristics of the culture medium. Furthermore, the stirring may be performed simultaneously with the cooling operation or immediately after the cooling operation. The stirring time is preferably 50 minutes or less, more preferably 40 minutes or less, and even more preferably 30 minutes or less, in order for the cells to detach from the temperature-responsive nonwoven fabric. In the case of small-scale culture where stirring is not possible, pipetting may be performed. 【0054】 The stirring speed in (2) and (II) above is not particularly limited as it depends on the cooling temperature and time, the amount of coolant, the shape and capacity of the culture vessel, the shape of the stirring blade, and the specific gravity of the nonwoven fabric, but as an example, 1 to 1000 rpm is preferred, 50 to 800 rpm is more preferred, and 100 to 500 rpm is even more preferred. 【0055】 In the steps of (3) and (III) above, where a new temperature-responsive nonwoven fabric is added to the culture medium, there are no particular limitations on the multiplier of the new temperature-responsive nonwoven fabric to be added. It can be determined according to the multiplier of scale-up in the expanded culture, for example, in the range of 1 to 100 times, preferably in the range of 1 to 50 times, and more preferably in the range of 4 to 20 times. 【0056】 In the step of adding new culture medium to the culture medium in (4) and (II) above, there are no particular limitations on the amount of new culture medium to be added, but it should be determined according to the culture scale for scaling up in expansion culture and the capacity of the bioreactor. For example, it is in the range of 1 to 100 times, preferably 1 to 50 times, and more preferably in the range of 4 to 20 times. 【0057】 For measuring the number of viable cells, it is recommended to first determine the viability of cells by staining the cytoplasm of dead cells blue using trypan blue, and then measure both the total number of cells and the number of viable cells. An automated cell counting device can be used for counting the number of cells. 【0058】 The method of this disclosure allows for highly efficient cell culture. Furthermore, according to one embodiment of this disclosure, cells can be cultured highly efficiently by adhering them to a temperature-responsive nonwoven fabric, culturing them until a predetermined number of cells are reached (for example, until confluent), then detaching the cells by cooling, and subsequently adding new temperature-responsive nonwoven fabric to perform subculturing (also called the cooled Fibers to Fibers Transfer method: cooled FtoF method). Alternatively, cells may be cultured on an expanded scale using the method of this disclosure. In this specification, expanded culture means increasing the volume of the culture environment. 【0059】 Using the method disclosed herein, subculturing can be performed on the same scale as the pre-culture, or on a different scale, for example, on a larger scale than the pre-culture. [Examples] 【0060】 Examples of the present disclosure are described below, but the present disclosure is not limited in any way by these examples. Unless otherwise specified, commercially available reagents were used. 【0061】 <1> Synthesis of Polymer 1 0.650 g (5 mmol) of 2-methoxyethyl acrylate (MEA) was added to a 200 mL two-necked flask, followed by 31.8 mg (100 μmol) of cyanomethyl dodecyl trithiocarbonate, 1.6 mg (10 μmol) of azobisisobutyronitrile, and 10 mL of tert-butyl alcohol. After purging with argon gas, the mixture was heated and stirred at 62 °C for 24 hours. 【0062】 After the first heating and stirring, 3.845 g (30 mmol) of n-butyl acrylate (BA) was added, followed by 1.6 mg (10 μmol) of azobisisobutyronitrile and 5 mL of tert-butyl alcohol. After purging with argon gas, the mixture was heated and stirred at 62°C for 24 hours. 【0063】 After the second heating and stirring, 7.355 g (65 mmol) of N-isopropylacrylamide (IPAAm) was added as the segment containing LCST, followed by 1.6 mg (10 μmol) of azobisisobutyronitrile and 85 mL of tert-butyl alcohol. After purging with argon gas, the mixture was heated and stirred at 62°C for 24 hours. 【0064】 After the third heating and stirring, the reaction mixture was reprecipitated and purified with water, and dried under reduced pressure to obtain a yellow solid. The obtained yellow solid was dissolved in chloroform, and the chloroform phase was recovered using a separatory funnel. The recovered chloroform phase was concentrated using an evaporator and reprecipitated and purified with heptane. The precipitate was recovered by filtration and dried under reduced pressure to obtain 8.295 g of polymer 1 (poly(MEA-BA-IPAAm)). The composition of the obtained polymer 1 was MEA:BA:IPAAm = 5:30:65 (mol%), the lower critical temperature limit was 32°C, and the number-average molecular weight Mn was 11.8 × 10⁻⁶. 4 The g / mol concentration and molecular weight distribution (Mw / Mn) were 1.45. 【0065】 <2> Synthesis of Polymer 2 1.156 g (8 mmol) of p-carboxystyrene (CSt, pKa = 4.20) and 1.271 g (12 mmol) of styrene (St, HLB value = 0) were added to a 100 mL two-necked flask. Then, 3.3 mg (20 μmol) of azobisisobutyronitrile and 20 mL of tert-butyl alcohol were added, and after purging with nitrogen gas, the mixture was heated and stirred at 64 °C for 24 hours. <1> The polymer 2 was purified using the same method as before, yielding 0.92 g of polymer 2 (poly(CSt / St)). The composition of the obtained polymer 2 was CSt:St = 33:67 (mol%), and the number-average molecular weight Mn was 10.6 × 10⁶. 4 The g / mol concentration and molecular weight distribution (Mw / Mn) were 1.84. 【0066】 <3> Polymer composition analysis The polymer composition was determined by proton nuclear magnetic resonance (1H-NMR) spectroscopy analysis using a nuclear magnetic resonance spectrometer (JEOL, product name JNM-ECZ400S / L1). 【0067】 <Analysis of molecular weight and molecular weight distribution of polymers> Weight-average molecular weight (Mw), number-average molecular weight (Mn), and molecular weight distribution (Mw / Mn) were measured by gel permeation chromatography (GPC). A Tosoh HLC-8320GPC was used as the GPC instrument, with two Tosoh TSKgel SuperAWM-H columns. The column temperature was set to 40°C, and the eluent was 2,2,2-trifluoroethanol containing 10 mM sodium trifluoroacetate. The sample was prepared at 1.0 mg / mL for measurement. A calibration curve for molecular weight was created using polymethyl methacrylate (Sigma-Aldrich), whose molecular weight is known. 【0068】 <4> Evaporator installation A Tokyo Rikakikai N-1300 rotary evaporator, a Tokyo Rikakikai CCA-1112A small cooling water circulation system, a Tokyo Rikakikai NVP-2100V diaphragm vacuum pump, and a Tokyo Rikakikai OSB-2200 water bath were installed. Water was used as the fluid for the small cooling water circulation system, and the temperature was set to 3°C. The water bath temperature was set to 50°C. 【0069】 <5> Preparation of polymer coating agents <1> 1.23 g of polymer 1 obtained from this, <2> The polymer 2 obtained was dissolved in 0.02 g and 48.75 g of 1-methoxy-2-propanol in a glass container by standing overnight. The mixture was then filtered through a 0.22 μm filter (Millipore, hydrophilic filter) to obtain the polymer coating agent. 【0070】 <6> Cell counting Ten nonwoven fabrics were collected after culture, and cells were separated and recovered from the fabrics by enzymatic treatment with trypsin-EDTA solution. The recovered cells were concentrated by centrifugation. After staining the concentrated cells with trypan blue, the cell count was measured using an automated cell counter (Countess3, Thermo Fisher Scientific, product number: AMQAX2000). 【0071】 <7> Counting of cumulative cell count The subculturing process was repeated three times according to the formula below, and the cumulative number of cells was counted after a total of 16 days of culture. 【0072】 First, by counting the number of cells in 10 nonwoven fabrics on the 4th day of culture, before the first subculturing, we calculated the number of cells recovered from one nonwoven fabric, as shown in [Equation 2]. [Math 2] The number of cells recovered from one nonwoven fabric sheet on day 4 of culture (cells / sheet) = the number of cells in 10 nonwoven fabric sheets on day 4 of culture (cells) × (1 nonwoven fabric sheet / 10 sheets) Next, by counting the cells from 10 nonwoven fabrics on day 8 of culture, before the second subculturing, the proliferation rate of the cells after the first subculturing was calculated as shown in [Equation 3]. [Math 3] Cell proliferation rate after the first subculturing = Number of cells recovered from one nonwoven fabric sheet on day 8 of culture (cells / sheet) / ((Number of seeded cells: Number of cells recovered from four nonwoven fabric sheets on day 4 of culture (cells)) / 40 nonwoven fabric sheets) Next, as shown in [Equation 4], the cumulative number of cells in the first passage was calculated. [Math 4] Cumulative cell count after first passage = Number of cells recovered from one nonwoven fabric sheet on day 4 of culture × 40 nonwoven fabric sheets × Cell proliferation rate after first passage Next, the number of cells in 10 nonwoven fabrics on day 12 of culture, before the second subculturing, was counted, and the cell proliferation ratio for the second subculturing was calculated following [Equation 3]. Then, the cumulative number of cells for the second subculturing was calculated as shown in [Equation 5]. [Number 5] Cumulative cell count after 2nd passage = Cumulative cell count after 1st passage (cells) × Cell proliferation rate after 2nd passage Then, the number of cells in 10 nonwoven fabrics on day 16 of culture, before the second subculturing, was counted, and the cell proliferation rate for the third subculturing was calculated following [Equation 3]. Finally, the cumulative number of cells for the third subculturing was calculated following [Equation 5]. 【0073】 Example 1: Preparation of temperature-responsive nonwoven fabric In a 200 mL flask <5> Add 0.1872 g of the polymer coating agent obtained, add 10 mL of methoxypropanol, and add nonwoven fabric (BioNOC2, manufactured by ESCO Bioengineering, specific surface area: 2400 cm²). 2 0.78 g (equivalent to 120 sheets) of polyethylene terephthalate (material: polyethylene terephthalate) was added and allowed to stand for 1 minute. The solvent was then removed by reducing the pressure using an evaporator to obtain a temperature-responsive nonwoven fabric. The nonwoven fabric used was 15.6 cm². 2 It is 0.0065g / sheet. 【0074】 Example 2: Subculture of Vero cells (cooled FtoF method) (1) Add 40 temperature-responsive nonwoven fabrics obtained in Example 1 to a 30 mL single-use bioreactor (Able Co., Ltd., product number: BWV-S03A), and add Vero cells (American Type Culture Collection (ATCC), product number: CCL-81) in a 2.50 × 10 4 Seeds were seeded at a cell density of cells per sheet. (2) The cultures were stirred and incubated at 60 rpm for 4 days in a 37°C incubator under a 5 vol% CO2 atmosphere. 30 mL of DMEM (Dulbecco's Modified Eagle Medium, manufactured by Wako Pure Chemical Industries) containing 10% fetal bovine serum (manufactured by BioWest) and 1% antibiotic-antifungal solution (manufactured by Wako Pure Chemical Industries) was used as the culture medium. (3) After stirring culture, the cell and temperature-responsive nonwoven fabric suspension was passed through a cell strainer with a mesh size of 70 μm (ASONE, product number: VCS-70) to remove suspended cells. (4) A new 30 mL single-use bioreactor was prepared, and four nonwoven fabrics to which the cells had adhered after step (3) were added, along with 36 new nonwoven fabrics. (5) A new culture medium cooled to 4°C was added to make a total volume of 10 mL, and the mixture was cooled by stirring at 100 rpm for 30 minutes at room temperature (23°C). (6) After cooling, 20 mL of fresh culture medium (37°C) was added, and the cells were cultured with stirring according to the method described in (2). In other words, the cells were subcultured in steps (3) to (6). (7) The subculturing process described in (3) to (6) was repeated three times, and the cells were cultured for a total of 16 days before the cell count was measured. 【0075】 The temperature-responsive nonwoven fabric disclosed herein allows cells to adhere once, and even after the cells have detached due to cooling to a temperature below LSCT, they can reattach if the temperature is raised to above LCST. This is because the nonwoven fabric itself can maintain its shape even with temperature changes. Furthermore, the temperature-responsive polymer introduced into the temperature-responsive nonwoven fabric does not dissolve into the culture medium when cooled, and exhibits hydrophobicity above LCST, meaning that the temperature response of the temperature-responsive nonwoven fabric is reversible. 【0076】 Comparative Example 1: Expansion culture of Vero cells (FtoF method) In Example 2(5), the same procedure as in Example 2 was followed, except that a new culture medium cooled to 4°C was replaced with a new medium at 37°C, and the number of cells after culturing was measured. 【0077】 Comparative Example 2 In Example 2, an untreated nonwoven fabric (BioNOC2, manufactured by ESCO Bioengineering, specific surface area: 2400 cm²) was used instead of the temperature-responsive nonwoven fabric. 2 The same procedure as in Example 2 was followed, except for the use of ( / g), and the number of cells after culturing was measured. 【0078】 Comparative Example 3 In the subculturing of Comparative Example 2, the same procedure as in Comparative Example 2 was performed except that a new medium cooled to 4°C was replaced with a new medium at 37°C, and the number of cells after culturing was measured. 【0079】 The culture results are shown in Tables 1-4 and Figure 1. It was found that cell culture can be efficiently performed by using temperature-responsive nonwoven fabric. Furthermore, it was found that cell culture can be efficiently performed by including a cooling step and agitation step in the cell culture process. In other words, it was found that cells can be efficiently cultured by using the method disclosed herein, in which cells are attached to temperature-responsive nonwoven fabric, cultured until a predetermined number of cells are reached, then the cells are detached by a cooling treatment, and then new temperature-responsive nonwoven fabric is added to perform subculturing (also called the cooled Fibers to Fibers Transfer method: cooled FtoF method). 【0080】 [Table 1] 【0081】 [Table 2] 【0082】 Table 3 【0083】 Table 4

Claims

[Claim 1] A method for culturing cells using a temperature-responsive nonwoven fabric containing a temperature-responsive polymer having a lower critical temperature (LCST) in the range of 0°C to 50°C, A step of culturing cells by adhering them to a temperature-responsive nonwoven fabric in a culture medium at a temperature of LCST or higher, The steps of cooling the temperature-responsive nonwoven fabric in the culture medium to below LCST and stirring or pipetting, A step of adding a new temperature-responsive nonwoven fabric to the culture medium, A step of adding a new culture medium to the culture medium, and The process includes a step of culturing the cells again by bringing the culture medium, after adding a new temperature-responsive nonwoven fabric and a new culture medium, to a temperature above LCST. A method wherein the temperature-responsive polymer is a block copolymer comprising the blocks (A), (B), and (C) below; (A) Polymer blocks with HLB values ​​(Griffin method) in the range of 7 to 20 (B) Polymer blocks with HLB values ​​(Griffin method) in the range of 0 or more and less than 7 (C) A temperature-responsive polymer block in which the LCST is in the range of 0°C to 50°C. [Claim 2] A method for culturing cells using a temperature-responsive nonwoven fabric containing a temperature-responsive polymer having an LCST in the range of 0°C to 50°C, A step of culturing cells by adhering them to a temperature-responsive nonwoven fabric in a culture medium at a temperature of LCST or higher, The process involves adding a new culture medium below LCST to the culture medium, thereby cooling the temperature-responsive nonwoven fabric to below LCST, and then stirring or pipetting it. A step of adding a new temperature-responsive nonwoven fabric to the culture medium, The process includes a step of culturing the cells again by bringing the culture medium, after adding a new culture medium and a new temperature-responsive nonwoven fabric, to a temperature of LCST or higher. A method wherein the temperature-responsive polymer is a block copolymer comprising the blocks (A), (B), and (C) below; (A) Polymer blocks with HLB values ​​(Griffin method) in the range of 7 to 20 (B) Polymer blocks with HLB values ​​(Griffin method) in the range of 0 or more and less than 7 (C) A temperature-responsive polymer block in which the LCST is in the range of 0°C to 50°C. [Claim 3] The method according to either claim 1 or 2, wherein the temperature-responsive polymer comprises a block polymer of 2-methoxyethyl acrylate, n-butyl acrylate, and N-isopropylacrylamide. [Claim 4] The steps of cooling the temperature-responsive nonwoven fabric in the culture medium to below LCST and stirring or pipetting, A step of adding a new temperature-responsive nonwoven fabric to the culture medium, A step of adding a new culture medium to the culture medium, and The method according to claim 1, wherein the step of culturing the cells again is repeated two or more times by bringing the culture medium, after adding a new temperature-responsive nonwoven fabric and a new culture medium, to a temperature of LCST or higher. [Claim 5] The process involves adding a new culture medium below LCST to the culture medium, thereby cooling the temperature-responsive nonwoven fabric to below LCST, and then stirring or pipetting it. A step of adding a new temperature-responsive nonwoven fabric to the culture medium, The method according to claim 2, wherein the step of culturing the cells again is repeated two or more times by bringing the culture medium, after adding a new culture medium and a new temperature-responsive nonwoven fabric, to a temperature of LCST or higher.

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