Expansion culture method using temperature-responsive microcarrier

Abstract

To provide an efficient culture method in a cell culture method employing a temperature-responsive microcarrier that is coated with a polymer showing a lower critical solution temperature.SOLUTION: The problem to be solved is solved by a cell culture method employing a temperature-responsive microcarrier that is coated with a polymer showing a lower critical solution temperature, the method including a cooling step and a stirring step in a subculture operation.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an expansion culture method using a temperature-responsive microcarrier. [Background technology]

[0002] In recent years, the market for cell-based biopharmaceuticals and regenerative medicine has grown significantly, creating a demand for technologies to efficiently mass-cultivate these cells. Cells are classified as either suspension cells or adherent cells, with most useful cells being the latter. Culturing adherent cells requires a scaffolding substrate, and plastic dishes and flasks have traditionally been used. However, as demand for cells expands, attention is now being paid to culture methods using microcarriers, which are suitable for mass cultivation.

[0003] The microcarrier culture method involves adding a suspension of cells and microcarriers to a medium, allowing the cells to adhere to the microcarrier surface, and then continuously stirring the culture medium to grow the cells attached to the microcarrier surface. Because the culture method using microcarriers is performed under agitation, it has the advantage of being able to achieve a higher cell density per unit volume than conventional static culture methods using dishes or flasks.

[0004] In conventional cell culture subculture methods using microcarriers, trypsin and other One method involves using a protease to detach cells from microcarriers, recovering the cell suspension, and adding it to a culture vessel containing new medium and new microcarriers (enzymatic method), while another method involves adding new microcarriers to the culture vessel containing the cultured microcarriers (beads-to-beads transfer method, or BtoB method). The former enzyme-based passaging method involves complex and time-consuming procedures, and the cells can be damaged by the enzyme treatment and recovery procedures. The latter passaging method relies on cells adhering to the microcarrier surface migrating to new microcarriers under agitation, resulting in non-uniform cell adhesion to the microcarriers and subsequent reduced cell proliferation rates. To address this issue, Patent Documents 1 and 2 disclose microcarriers coated with a polymer exhibiting a lower critical temperature. According to these documents, cooling induces a sol transition of the polymer exhibiting a lower critical temperature, weakening the adhesive force on the microcarrier surface and allowing cells to detach from the microcarrier. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6954047 [Patent Document 2] JP 2021-106543 A Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a highly efficient cell culture method using a temperature-responsive microcarrier coated with a polymer that exhibits a lower critical temperature. [Means for solving the problem]

[0007] In view of the above, the present inventors have conducted extensive research and discovered that cells can be cultured with high efficiency by a cell culture method using temperature-responsive microcarriers coated with a polymer that exhibits a lower critical temperature, which method includes a cooling step and a stirring step in the passaging operation (cooled Beads to Beads Transfer method: cooled BtoB method), and have completed the present invention. That is, the present invention encompasses the following aspects.

[0008] [1] A method for culturing adherent cells using a temperature-responsive microcarrier coated with a polymer exhibiting a lower critical temperature, comprising: (1) culturing adherent cells on a temperature-responsive microcarrier surface in a culture medium at a temperature equal to or higher than the lower critical temperature; (2) cooling the temperature-responsive microcarriers in the culture solution to a temperature equal to or lower than the lower critical temperature and stirring; (3) adding new temperature-responsive microcarriers to the culture solution; (4) adding fresh complete medium and / or fresh basal medium to the culture medium; (5) culturing the cells again by raising the temperature of the culture medium to a temperature equal to or higher than the lower critical temperature; wherein steps (2) to (4) are carried out in any order.

[0009] [2] The method according to [1], wherein the stirring time in step (2) is 50 minutes or less.

[0010] [3] The method according to [1] or [2], which comprises repeating the steps (1) to (5) two or more times.

[0011] [4] The method according to any one of [1] to [3], wherein the specific gravity of the temperature-responsive microcarrier is in the range of 1.01 to 2.00.

[0012] [5] The method according to any one of [1] to [4], wherein the lower critical temperature of the temperature-responsive microcarrier is 0°C to 50°C.

[0013] [6] The method according to any one of [1] to [5], selected from the following (I) or (II): (I) when the step (2) is a cooling operation using a culture medium in a culture vessel, it is a method of removing 0 (v / v) % to 90 (v / v) % of the culture medium in the culture vessel and lowering the temperature of the remaining culture medium to a temperature not higher than the lower critical temperature, and the step (4) is a step of using a new completed medium and / or a new basal medium whose temperature is not lower than the lower critical temperature; (II) When step (2) is a cooling operation using a new complete medium and / or a new basal medium, a step of adding a new complete medium and / or a new basal medium cooled to or below the lower critical temperature and lowering the temperature in the culture vessel to or below the lower critical temperature. [Effects of the Invention]

[0014] In a cell culture method using a temperature-responsive microcarrier coated with a polymer exhibiting a lower critical temperature, highly efficient culture can be achieved by including a cooling step and a stirring step in the subculture procedure. Therefore, the present invention provides a method that enables efficient expansion culture and is useful for industrial culture of adherent cells. [Brief explanation of the drawings]

[0015] [Figure 1] A graph comparing the number of hBMSC cells depending on the microcarrier and passage method. [Figure 2] A diagram comparing the cell count of MDCK cells depending on the microcarrier and passage method. [Figure 3] A diagram comparing the cell count of Vero cells depending on the microcarrier and passage method. [Figure 4] A diagram comparing the cell count of BHK-21 cells depending on the microcarrier and passaging method. [Figure 5] A diagram comparing the cell count of Vero cells depending on the microcarrier and passage method. [Figure 6] A diagram comparing the cell count of BHK-21 cells depending on the microcarrier and passaging method. DETAILED DESCRIPTION OF THE INVENTION

[0016] Hereinafter, the embodiments of the present invention will be described in detail, but the present invention is not intended to be limited to the following contents. The present invention can be practiced with appropriate modifications within the scope of its spirit.

[0017] The present specification provides a method for culturing adherent cells using a temperature-responsive microcarrier coated with a polymer that exhibits a lower critical temperature, comprising the steps of: (1) culturing the cells on the surface of the temperature-responsive microcarrier in a culture medium at a temperature equal to or higher than the lower critical temperature; (2) cooling the temperature-responsive microcarriers in the culture solution to a temperature equal to or lower than the lower critical temperature and stirring; (3) adding new temperature-responsive microcarriers to the culture solution; (4) adding fresh complete medium and / or fresh basal medium to the culture medium; (5) culturing the cells again by raising the temperature of the culture medium to a temperature equal to or higher than the lower critical temperature; wherein steps (2) to (4) are performed in any order.

[0018] The lower critical solution temperature (LCST, hereinafter sometimes referred to as LCST) is the temperature below which a polymer dissolves in water to form a transparent solution, but above which it becomes insoluble and cloudy, or precipitates, resulting in 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 25°C to 35°C.

[0019] Examples of repeating units of homopolymers exhibiting lower critical temperatures and their lower critical temperatures 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), and N-methyl-Nn-propylacrylamide (LCST = 20°C). The temperature at which the lower critical temperature appears varies depending on the concentration of the aqueous solution, but N-isopropylacrylamide is preferred because the appearance of the lower critical temperature is less dependent on the concentration.

[0020] The repeating unit of the polymer exhibiting the lower critical temperature in the present invention may be of one type only, or may be a combination of two or more types. Furthermore, as long as the polymer has an LCST, it may contain a repeating unit of a polymer that does not exhibit an LCST in addition to a repeating unit of a polymer that exhibits an LCST, depending on the purpose, such as improving adhesiveness to cells or microcarriers. For example, the composition of N-isopropylacrylamide exhibiting an LCST in the polymer is 10 mol% to 95 mol%, preferably 30 mol% to 80 mol%, and more preferably 65 mol% to 70 mol%.

[0021] In addition to the polymer exhibiting LCST, other polymer compounds may be introduced depending on the purpose, such as improving adhesiveness to cells or microcarriers. For example, in the examples of the present invention, a polymer compound consisting of carboxystyrene and styrene is introduced to improve adhesiveness to cells.

[0022] The material of the microcarrier is not particularly limited, but examples include polystyrene, polymethyl methacrylate, polyethylene terephthalate, polycarbonate, cellulose, cyclodextrin, acrylamide, alginate, dextran, gelatin, glass, and mixtures of two or more of these.

[0023] The physical properties of the microcarriers are not particularly limited, but the specific gravity is 1.01 to 2.00 so that the microcarriers will settle in the culture medium, and preferably 1.01 to 1.30, more preferably 1.01 to 1.10 so that they can be easily dispersed under stirring.

[0024] The shape of the microcarrier is not particularly limited, but examples include spheres, ellipsoids, plates, and tubes. Furthermore, the microcarrier may or may not be porous. If porous, there is no limitation on the pore size.

[0025] The diameter of the microcarrier is not particularly limited, but since cells are cultured on the surface of the beads, it is preferable that it is larger than the cells to be cultured, and the major axis of the microcarrier is preferably 20 μm to 1000 μm, more preferably 20 μm to 700 μm, and even more preferably 50 μm to 400 μm. If the major axis is smaller than the above range, separation from the cells becomes difficult and the recovery rate decreases. Furthermore, if the major axis is larger than the above range, the culture area per volume becomes smaller.

[0026] As used herein, the term "temperature-responsive microcarrier" refers to a microcarrier coated with a polymer that exhibits a lower critical temperature.

[0027] Cultured cells can be detached from the temperature-responsive microcarrier of the present invention by adjusting the temperature to a level below the LCST of the polymer constituting the temperature-responsive layer. Detachment by temperature change may be performed in the culture medium or by adding new medium. Furthermore, stirring is preferred to further improve cell detachment during temperature response. Stirring may be performed simultaneously with 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 terms of detaching cells from the temperature-responsive microcarrier.

[0028] There are no particular limitations on the method for coating the microcarrier with a polymer that exhibits a lower critical temperature. Examples include a method in which a repeating unit that exhibits a lower critical temperature is chemically coated by electron beam irradiation, and a method in which a surface treatment agent in which a polymer that exhibits a lower critical temperature is dissolved in a solvent is applied to the microcarrier to physically coat it.

[0029] The surface of a microcarrier coated with a polymer exhibiting LCST may be further coated with an extracellular matrix. The type of extracellular matrix is ​​not particularly limited, and examples include collagen, atelocollagen, hyaluronic acid, elastin, proteoglycan, glycosaminoglycan, fibronectin, laminin, vitronectin, gelatin, and Matrigel, which contains laminin, collagen IV, heparan sulfate proteoglycan, entactin / nidogen 1, 2, or the like as its main components. These may be used alone or in combination of two or more types. Alternatively, the extracellular matrix may be a segment of these extracellular matrices.

[0030] Adherent cells are cells that grow while adhering to the surface of a cell culture substrate such as a microcarrier. The origin of the cells is not particularly limited, and 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 examples of cells include various cultured cell lines such as 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, as well as epithelial and endothelial cells that constitute various tissues and organs in vivo. Examples of cells include contractile skeletal muscle cells, smooth muscle cells, cardiac muscle cells, neuronal cells that make up the nervous system, glial cells, and fibroblasts, macrophages and dendritic cells involved in the body's immune system, hepatic parenchymal cells, non-hepatic parenchymal cells, and adipocytes that are involved in the body's metabolism, and cells with differentiation potential include various stem cells 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, and germline stem cells, as well as progenitor cells of various tissues, and cells induced to differentiate from these. Mesenchymal stem cells are particularly preferred. Mesenchymal stem cells refer to a population of stem cells and their progenitor cells that can differentiate into all or some of the mesenchymal cell types, such as chondrocytes, osteoblasts, and adipocytes. The origin of mesenchymal stem cells is not particularly limited, but examples include tissues such as bone marrow, adipose tissue, dental pulp, umbilical cord blood, placenta, and synovium, as well as pluripotent stem cells such as ES cells and iPS cells. In addition to the above-mentioned cells, cells that have been appropriately genetically modified depending on the purpose may also be used.

[0031] In step (1), the temperature of the culture medium in the culture vessel is kept at or above the lower critical temperature, so that the polymer exhibiting the lower critical temperature gels, allowing the adherent cells to adhere to and proliferate on the microcarrier. While there are no limitations on the temperature for cell culture, for example, when using mammalian cells such as human or other animals, it is recommended to culture the cells at temperatures close to body temperature to achieve 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. Conditions other than the lower critical temperature are not particularly limited; for example, either static culture or agitation culture may be used, but agitation culture is preferred, as it allows for a larger culture area per unit volume.

[0032] The method of the present invention enables highly efficient expansion of adherent cells. In the present invention, expansion refers to increasing the volume of the culture environment. Expansion culture using the method of the present invention can be performed using microcarriers to which cells are attached, either in static or suspension culture. However, suspension culture is preferred because it allows for favorable dispersion of nutrients in the medium and cell waste products. Suspension culture can be performed using either shaking or stirring culture, but stirring culture is preferred due to its utility on an industrial scale. The stirring speed required to disperse microcarriers in the culture medium depends on the shape and volume of the culture vessel, the shape of the stirring blades, and the specific gravity of the microcarriers. For example, in the case of a 1-50 L stirred tank bioreactor, it is preferred to perform the culture at 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.

[0033] The concentration of microcarriers in the culture solution can be adjusted appropriately based on the culture cell type, cell seeding density, stirring speed, and the size and specific gravity of the microcarriers, and can be, for example, 0.01 to 100 g / L, 0.1 to 50 g / L, 0.5 to 20 g / L, or 1 to 10 g / L.

[0034] There is no particular limitation on the cell culture density as long as the cells adhere and proliferate. In the case of human-derived mesenchymal stem cells, for example, 1.0 × 10 per surface area of ​​the microcarrier is used. 1 cells / cm 2 ~1.0×10 5 cells / cm 2 is preferred, and 1.0 × 10 2 cells / cm 2 ~1.0×10 4 cells / cm 2 In the case of Vero cells, for example, 1.0 x 10 per surface area of ​​the microcarrier is more preferred. 1 cells / cm 2 ~1.0×10 5 cells / cm 2 is preferred, and 5.0 × 10 2 cells / cm 2 ~5.0×10 3 cells / cm 2 Other culture conditions are not particularly limited, and the culture may be carried out under conditions commonly used in the art.

[0035] In the present invention, the cell culture vessel is not particularly limited as long as it can agitate and suspend microcarriers. The cell culture vessel 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.

[0036] The type of basal medium used in the culture of the present invention is not particularly limited, and examples that can be used include MEM, αMEM, DMEM, EMEM, GMEM, DMEM / Ham's F-12, Ham's F-12, Ham's F-10, Medium 199, RPMI 1640, etc. Furthermore, the basal medium can be appropriately selected and used depending on the type of cell.

[0037] The composition of the complete medium used in the culture of the present invention is not particularly limited, and it may be a serum medium in which serum is added to the above-mentioned basal medium, or a serum-free medium that does not contain serum. However, the serum used is not particularly limited, and examples that can be used include fetal bovine serum (FBS), calf serum, adult bovine serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rabbit serum, and human serum. Furthermore, the complete medium may or may not contain antibiotics. Furthermore, additives other than serum and antibiotics can be selected appropriately and added at appropriate concentrations depending on the type of cell.

[0038] A known method for measuring cell viability is to determine whether cells are viable or dead using trypan blue staining, which stains the cytoplasm of dead cells blue, and then measure the total number of cells and the number of live cells.An example of a method for counting cells is to use an automated cell measuring device. [Example]

[0039] Examples of the present invention will be described below, but the present invention is not limited to these examples. Unless otherwise specified, commercially available reagents were used.

[0040] Example 1 Expansion of bone marrow-derived human mesenchymal stem cells (hBMSCs) <1> Polymer synthesis 0.650 g (5 mmol) of 2-methoxyethyl acrylate (MEA) was added to a 200 mL two-neck 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 replacing the atmosphere with argon gas, the mixture was heated and stirred at 62°C for 24 hours.

[0041] 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 argon gas replacement, the mixture was heated and stirred at 62° C. for 24 hours.

[0042] After the second heating and stirring, 7.355 g (65 mmol) of N-isopropylacrylamide (IPAAm) was added as a segment having an LCST to the above, and then 1.6 mg (10 μmol) of azobisisobutyronitrile and 85 mL of tert-butyl alcohol were added. After replacing the atmosphere with argon gas, the mixture was heated and stirred at 62°C for 24 hours.

[0043] After the third heating and stirring, the reaction solution was purified by reprecipitation 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 purified by reprecipitation with heptane. The precipitate was recovered by filtration and dried under reduced pressure to obtain 8.295 g of polymer poly(MEA-BA-IPAAm). The composition of the obtained polymer was MEA:BA:IPAAm = 5:30:65 (mol%), the lower critical temperature was 32°C, and the number average molecular weight Mn was 11.8 x 10 4 g / mol, and the molecular weight distribution Mw / Mn was 1.45.

[0044] <2> Synthesis of polymer compounds A 100 mL two-neck flask was charged with 1.156 g (8 mmol) of p-carboxystyrene (CSt, pKa = 4.20) and 1.271 g (12 mmol) of styrene (St, HLB = 0). 3.3 mg (20 μmol) of azobisisobutyronitrile and 20 mL of tert-butyl alcohol were then added, and the mixture was purged with nitrogen gas and heated and stirred at 64°C for 24 hours. <1> The resulting polymer compound had a composition of CSt:St=33:67 (mol%) and a number-average molecular weight Mn of 10.6×10 4 The molecular weight distribution Mw / Mn was 1.84.

[0045] <Polymer composition analysis> The values ​​were determined by proton nuclear magnetic resonance spectroscopy (1H-NMR) using a nuclear magnetic resonance analyzer (manufactured by JEOL Ltd., trade name JNM-ECZ400S / L1).

[0046] <Analysis of polymer molecular weight and molecular weight distribution> 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 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 adjusted to 1.0 mg / mL. A molecular weight calibration curve was prepared using polymethyl methacrylate (Sigma-Aldrich) of known molecular weight.

[0047] <3> Preparation of surface treatment agent 1 <1> 1.00 g of the polymer obtained in <2> 0.0075 g of the polymer compound obtained in 2. and 48.9925 g of 1-methoxy-2-propanol were placed in a glass container and left to stand overnight to dissolve. The mixture was then filtered through a 0.22 μm filter (manufactured by Millipore, hydrophilic filter) to obtain surface treatment agent 1.

[0048] <4> Preparation of temperature-responsive microcarriers 1 In a 200 mL eggplant flask, 10 g of AmberChrom 1x8 chloride foam 100-200 mesh (Sigma-Aldrich, product number: 217425, average particle size: 130 μm, specific gravity: 1.08, hereinafter also referred to as "MC5") was added. <3> 1.06 g of the surface treatment agent 1 obtained in 1. and 15 g of 1-methoxy-2-propanol were added and left to stand for 1 hour. The solvent was then removed by reducing the pressure in an evaporator to obtain temperature-responsive microcarrier 1 (hereinafter also referred to as "temperature-responsive MC1").

[0049] <5> Culture of bone marrow-derived human mesenchymal stem cells (hBMSCs) 0.2 g of temperature-responsive MC1 was added to a 30 mL single-use bioreactor (Able, product number: BWV-S03A), and 4.00 × 10 bone marrow-derived human mesenchymal stem cells (Lonza Japan, product number: PT-2501, Lot Number: 0000603525) were added.3 cells / cm 2 The cells were seeded at a cell density of 100 rpm. Agitation culture was performed for 6 days in a 37°C incubator with a 5 vol% CO2 atmosphere at 100 rpm. 30 mL of Mesenchymal Stem Cell Growth Medium 2 (PromoCell, product number: C-28009) was used as the medium. The agitation speed was gradually increased from 0 rpm until the microcarriers no longer accumulated at the bottom of the tank.

[0050] <6> Cell passaging (i) After stirring culture, 10 mL of the cell and microcarrier suspension was passed through a cell strainer with 70 μm openings (manufactured by ASONE, product number: VCS-70) to remove floating cells. (ii) Only the microcarriers with cells attached were collected, and the cells were separated and collected from the microcarriers by enzymatic treatment with trypsin-EDTA solution. The collected cells were concentrated by centrifugation, stained with trypan blue, and then counted using a Countess 3 automated cell counter (Thermo Fisher Scientific, product number: AMQAX2000). (iii) Prepare a new 30 mL single-use bioreactor; <5> The microcarriers with the cells attached in (i) were added to the medium so that the cell density was as described in (1), and new microcarriers were added so that the total amount was 0.2 g. New medium cooled to 4°C was added to the medium so that the volume was 30 mL, and the mixture was cooled by stirring at 100 rpm at room temperature (23°C) for 30 minutes. (iv) After cooling treatment, <5> Spinner culture was carried out according to the method described in .

[0051] <7> <6> After repeating the subculture three times and culturing for a total of 24 days, <6> The procedures described in (i) and (ii) were carried out, and the cell number was measured.

[0052] Comparative Example 1 Example 1 <6> In (iii), the same procedure as in Example 1 was carried out except that fresh medium at 37°C was added instead of fresh medium cooled to 4°C, and the number of cells after culture was measured.

[0053] Comparative Example 2 The same procedure as in Example 1 was carried out except that MC5, which is not temperature responsive, was used instead of the temperature responsive MC1, and the number of cells after culture was measured.

[0054] Comparative Example 3 The same procedure as in Comparative Example 2 was carried out except that in the subculture in Comparative Example 2, fresh medium at 37°C was added instead of fresh medium cooled to 4°C, and the number of cells after culture was measured.

[0055] Comparative Example 4 In Example 1, the same procedures as in Example 1 were performed except that a non-temperature-responsive CellBIND (registered trademark) surface microcarrier (manufactured by Corning, specific gravity: 1.026, hereinafter also referred to as "MC6") was used instead of the temperature-responsive MC1, and the stirring speed during culture was changed from 100 rpm to 60 rpm to match the specific gravity of the microcarrier, and the number of cells after culture was measured.

[0056] Comparative Example 5 The same procedure as in Comparative Example 4 was carried out except that in the subculture in Comparative Example 4, fresh medium at 37°C was added instead of fresh medium cooled to 4°C, and the number of cells after culture was measured.

[0057] The results of Example 1 and Comparative Examples 1 to 5 are shown in FIG.

[0058] Example 2 Expansion of MDCK cells <1> Example 1 <1> ~ <4> The above procedure was carried out to obtain temperature-responsive MC1.

[0059] <2> MDCK cell culture 0.2 g of temperature-responsive MC1 was added to a 30 mL single-use bioreactor (Able, product number: BWV-S03A), and 8.50 × 10 MDCK cells (JCRB Cell Bank, product number: JCRB9029) were added. 3 cells / cm 2The cells were seeded at a cell density of 100 rpm for 7 days in a 37°C incubator with a 5 vol% CO2 atmosphere. The medium used was 30 mL of DMEM (Dulbecco's Modified Eagle Medium, Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum (BioWest) and 1% antibiotic-antimycotic solution (Wako Pure Chemical Industries, Ltd.).

[0060] <3> Cell passaging (i) After stirring culture, 10 mL of the cell and microcarrier suspension was passed through a cell strainer with 70 μm openings (manufactured by ASONE, product number: VCS-70) to remove floating cells. (ii) Only the microcarriers with cells attached were collected, and the cells were separated and collected from the microcarriers by enzymatic treatment with trypsin-EDTA solution. The collected cells were concentrated by centrifugation, stained with trypan blue, and then counted using a Countess 3 automated cell counter (Thermo Fisher Scientific, product number: AMQAX2000). (iii) Prepare a new 30 mL single-use bioreactor; <2> The microcarriers with the cells attached in (i) were added to the medium so that the cell density was as described in (1), and new microcarriers were added so that the total amount was 0.2 g. New medium cooled to 4°C was added to the medium so that the volume was 30 mL, and the mixture was cooled by stirring at 100 rpm at room temperature (23°C) for 30 minutes. (iv) After cooling treatment, Example 2 <2> Spinner culture was carried out according to the method described in .

[0061] <4> <3> After repeating the subculture three times and culturing for a total of 28 days, <3> The procedures described in (i) and (ii) were carried out, and the cell number was measured.

[0062] Comparative Example 6 Example 2 <3> In (iii), the same procedure as in Example 2 was carried out except that fresh medium at 37°C was added instead of fresh medium cooled to 4°C, and the number of cells after culture was measured.

[0063] Comparative Example 7 The same procedure as in Example 2 was carried out except that MC5, which is not temperature responsive, was used instead of the temperature responsive MC1, and the number of cells after culture was measured.

[0064] Comparative Example 8 The same procedure as in Comparative Example 7 was carried out except that in the subculture in Comparative Example 7, fresh medium at 37°C was added instead of fresh medium cooled to 4°C, and the number of cells after culture was measured.

[0065] Comparative Example 9 In Example 2, the same procedure as in Example 2 was performed, except that non-temperature-responsive MC6 was used instead of temperature-responsive MC1, and the stirring speed during culture was changed from 100 rpm to 60 rpm to match the specific gravity of the microcarrier, and the number of cells after culture was measured.

[0066] Comparative Example 10 The same procedure as in Comparative Example 9 was carried out except that in the subculture in Comparative Example 9, fresh medium at 37°C was added instead of fresh medium cooled to 4°C, and the number of cells after culture was measured.

[0067] The results of Example 2 and Comparative Examples 6 to 10 are shown in FIG. Example 3 Expansion of Vero cells <1> Example 1 <1> ~ <4> The above procedure was carried out to obtain temperature-responsive MC1.

[0068] <2> Vero cell culture 0.2 g of temperature-responsive MC1 was added to a 30 mL single-use bioreactor (Able, product number: BWV-S03A), and 6.50 × 10 Vero cells (American Type Culture Collection (ATCC), product number: CCL-81) were added. 3 cells / cm 2The cells were seeded at a cell density of 100 rpm for 7 days in a 37°C incubator with a 5 vol% CO2 atmosphere. The medium used was 30 mL of DMEM (Dulbecco's Modified Eagle Medium, Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum (BioWest) and 1% antibiotic-antimycotic solution (Wako Pure Chemical Industries, Ltd.).

[0069] <3> Cell passaging (i) After stirring culture, 10 mL of the cell and microcarrier suspension was passed through a cell strainer with 70 μm openings (manufactured by ASONE, product number: VCS-70) to remove floating cells. (ii) Only the microcarriers with cells attached were collected, and the cells were separated and collected from the microcarriers by enzymatic treatment with trypsin-EDTA solution. The collected cells were concentrated by centrifugation, stained with trypan blue, and then counted using a Countess 3 automated cell counter (Thermo Fisher Scientific, product number: AMQAX2000). (iii) Prepare a new 30 mL single-use bioreactor; <2> The microcarriers with the cells attached in (i) were added to the medium so that the cell density was as described in (1), and new microcarriers were added so that the total amount was 0.2 g. New medium cooled to 4°C was added to the medium so that the volume was 30 mL, and the mixture was cooled by stirring at 100 rpm at room temperature (23°C) for 30 minutes. (iv) After cooling treatment, <2> Spinner culture was carried out according to the method described in .

[0070] <4> <3> After repeating the subculture three times and culturing for a total of 28 days, <3> The procedures described in (i) and (ii) were carried out, and the cell number was measured.

[0071] Comparative Example 11 Example 3 <3> In (iii), the same procedure as in Example 3 was carried out except that fresh medium at 37°C was added instead of fresh medium cooled to 4°C, and the number of cells after culture was measured.

[0072] Comparative Example 12 The same procedure as in Example 3 was carried out except that MC5, which is not temperature responsive, was used instead of the temperature responsive MC1, and the number of cells after culture was measured.

[0073] Comparative Example 13 The same procedure as in Comparative Example 12 was carried out except that in the subculture in Comparative Example 12, fresh medium at 37°C was added instead of fresh medium cooled to 4°C, and the number of cells after culture was measured.

[0074] Comparative Example 14 In Example 3, the same procedure as in Example 2 was performed except that non-temperature-responsive MC6 was used instead of temperature-responsive MC1, and the stirring speed during culture was changed from 100 rpm to 60 rpm to match the specific gravity of the microcarriers, and the number of cells after culture was measured.

[0075] Comparative Example 15 The same procedure as in Comparative Example 14 was carried out except that in the subculture in Comparative Example 14, fresh medium at 37°C was added instead of fresh medium cooled to 4°C, and the number of cells after culture was measured.

[0076] The results of Example 3 and Comparative Examples 11 to 15 are shown in FIG. Example 4 Expansion of BHK-21 cells <1> Example 1 <1> ~ <3> The above procedure was carried out to obtain surface treatment agent 1.

[0077] <2> Preparation of temperature-responsive microcarriers 2 In a 200 mL eggplant flask, 10 g of polystyrene microcarriers (average particle size: 130 μm, specific gravity: 1.03, hereinafter also referred to as "MC7") were placed. <1> 1.06 g of the surface treatment agent 1 obtained in 1. and 15 g of 1-methoxy-2-propanol were added and left to stand for 1 hour. The solvent was then removed by reducing the pressure in an evaporator to obtain temperature-responsive microcarrier 2 (hereinafter also referred to as "temperature-responsive MC2").

[0078] <3> Culture of BHK-21 cells 0.2 g of temperature-responsive MC2 was added to a 30 mL single-use bioreactor (Able, product number: BWV-S03A), and 6.50 × 10 BHK-21 cells (ATCC, product number: CCL-10) were added. 3 cells / cm 2 The cells were seeded at a cell density of 1000 kJ / ml. Agitation culture was performed for 7 days at 60 rpm in a 37°C incubator with a 5 vol% CO2 atmosphere. The medium used was 30 mL of DMEM (Dulbecco's Modified Eagle Medium, manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum (manufactured by BioWest) and 1% antibiotic-antimycotic solution (manufactured by Wako Pure Chemical Industries, Ltd.).

[0079] <4> Cell passaging (i) After stirring culture, 10 mL of the cell and microcarrier suspension was passed through a cell strainer with 70 μm openings (manufactured by ASONE, product number: VCS-70) to remove floating cells. (ii) Only the microcarriers with cells attached were collected, and the cells were separated and collected from the microcarriers by enzymatic treatment with trypsin-EDTA solution. The collected cells were concentrated by centrifugation, stained with trypan blue, and then counted using a Countess 3 automated cell counter (Thermo Fisher Scientific, product number: AMQAX2000). (iii) Prepare a new 30 mL single-use bioreactor; <3> The microcarriers with the cells attached in (i) were added to the medium so that the cell density was as described in (1), and new microcarriers were added so that the total volume was 0.2 g. New medium cooled to 4°C was added to the medium so that the volume was 30 mL, and the mixture was cooled by stirring at 100 rpm at room temperature (23°C) for 30 minutes. (iv) After cooling treatment, <3> Spinner culture was carried out according to the method described in .

[0080] <5> <4> After repeating the subculture three times and culturing for a total of 28 days, <4> The procedures described in (i) and (ii) were carried out, and the cell number was measured.

[0081] Comparative Example 16 Example 4 <4> In (iii), the same procedure as in Example 4 was carried out except that fresh medium at 37°C was added instead of fresh medium cooled to 4°C, and the number of cells after culture was measured.

[0082] Comparative Example 17 The same procedure as in Example 4 was carried out except that MC6, which is not temperature responsive, was used instead of the temperature responsive MC2, and the number of cells after culture was measured.

[0083] Comparative Example 18 The same procedure as in Comparative Example 17 was carried out except that in the subculture in Comparative Example 17, fresh medium at 37°C was added instead of fresh medium cooled to 4°C, and the number of cells after culture was measured.

[0084] Comparative Example 19 The same procedure as in Example 4 was carried out except that MC7, which is not temperature responsive, was used instead of the temperature responsive MC2, and the number of cells after culture was measured.

[0085] Comparative Example 20 The same procedure as in Comparative Example 19 was carried out except that in the subculture in Comparative Example 19, fresh medium at 37°C was added instead of fresh medium cooled to 4°C, and the number of cells after culture was measured.

[0086] The results of Example 4 and Comparative Examples 16 to 20 are shown in FIG.

[0087] Example 5 Expansion of Vero cells <1> Example 1 <1> ~ <2> The above operations were carried out to obtain polymers and polymer compounds.

[0088] <2> Preparation of surface treatment agent 2 <1> 1.00 g of the polymer obtained in 1. above, 0.0300 g of a polymer compound, and 48.9700 g of 1-methoxy-2-propanol were placed in a glass container and left to stand overnight to dissolve. The mixture was then filtered through a 0.22 μm filter (manufactured by Millipore, hydrophilic filter) to obtain surface treatment agent 2.

[0089] <3> 3. Preparation of Temperature-Responsive Microcarriers 10g of MC5 in a 200mL eggplant flask, <2> 1.06 g of the surface treatment agent 2 obtained in 1. and 15 g of 1-methoxy-2-propanol were added and left to stand for 1 hour. The solvent was then removed by reducing the pressure in an evaporator to obtain temperature-responsive microcarrier 3 (hereinafter also referred to as "temperature-responsive MC3").

[0090] <4> Example 3 <2> ~ <4> The same procedure as in Example 3 was repeated except that temperature-responsive MC3 was used instead of temperature-responsive MC1. <2> ~ <4> The same procedure as in the above was carried out, and the number of cells after culture was measured.

[0091] Comparative Example 21 The same procedure as in Example 5 was carried out except that in the subculture in Example 5, fresh medium at 37°C was added instead of fresh medium cooled to 4°C, and the number of cells after culture was measured.

[0092] The results of Example 5 and Comparative Examples 12 to 15 and 21 are shown in FIG.

[0093] Example 6 Expansion of BHK-21 cells <1> Example 5 <1> ~ <2> The above procedure was carried out to obtain surface treatment agent 2.

[0094] <2> 4. Preparation of Temperature-Responsive Microcarriers 10g of MC7 in a 200mL eggplant flask, <1> 1.06 g of the surface treatment agent 2 obtained in 1. and 15 g of 1-methoxy-2-propanol were added and left to stand for 1 hour. The solvent was then removed by reducing the pressure in an evaporator to obtain temperature-responsive microcarrier 4 (hereinafter also referred to as "temperature-responsive MC4").

[0095] <3> Example 4 <3> ~ <5> The same procedure as in Example 4 was repeated except that temperature-responsive MC4 was used instead of temperature-responsive MC2. <3> ~ <5> The same procedure as in the above was carried out, and the number of cells after culture was measured.

[0096] Comparative Example 22 The same procedure as in Example 6 was carried out except that in the subculture in Example 6, fresh medium at 37°C was added instead of fresh medium cooled to 4°C, and the number of cells after culture was measured.

[0097] The results of Example 6 and Comparative Examples 17 to 20 and 22 are shown in FIG.

[0098] 1 to 6 show that the use of temperature-responsive microcarriers increases the number of cells after expansion culture, and that the effect becomes more pronounced when a cooling treatment is included in the subculture step.

Claims

1. A method for culturing adherent cells using a temperature-responsive microcarrier coated with a polymer exhibiting a lower critical temperature, comprising: (1) culturing adherent cells on a temperature-responsive microcarrier surface in a culture medium at a temperature equal to or higher than the lower critical temperature; (2) cooling the temperature-responsive microcarriers in the culture solution to a temperature equal to or lower than the lower critical temperature and stirring; (3) adding new temperature-responsive microcarriers to the culture solution; (4) adding fresh complete medium and / or fresh basal medium to the culture; (5) culturing the cells again by heating the culture medium to a temperature equal to or higher than the lower critical temperature; wherein steps (2) to (4) are performed in any order.

2. The method according to claim 1, wherein the stirring time in step (2) is 50 minutes or less.

3. The method according to claim 1 or 2, comprising repeating steps (1) to (5) two or more times.

4. The method according to any one of claims 1 to 3, wherein the specific gravity of the temperature-responsive microcarrier is in the range of 1.01 to 2.

00.

5. The method according to any one of claims 1 to 3, wherein the lower critical temperature of the temperature-responsive microcarrier is 0°C to 50°C.

6. The method according to claims 1 to 3, wherein the compound is selected from the following (I) or (II): (I) when the step (2) is a cooling operation using a culture medium in a culture vessel, it is a method of removing 0 (v / v) % to 90 (v / v) % of the culture medium in the culture vessel and lowering the temperature of the remaining culture medium to a temperature not higher than the lower critical temperature, and the step (4) is a step of using a new completed medium and / or a new basal medium whose temperature is not lower than the lower critical temperature; (II) When the step (2) is a cooling operation using a new complete medium and / or a new basal medium, a step of adding a new complete medium and / or a new basal medium cooled to below the lower critical temperature and lowering the temperature in the culture vessel to below the lower critical temperature.

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