Cell culture method

Abstract

The present invention provides a cell culture method that comprises: a first step for preparing a cell mass population having a long diameter of 400 μm or less; and a second step for suspension culturing the cell mass population obtained in the first step.

Description

[Technical Field]

[0001] The present invention relates to a cell culture method, and more particularly to a cell culture method including a step of controlling the size of cell clusters. [Background technology]

[0002] Regenerative medicine techniques using cells have attracted much attention in recent years as a potential means of treating various diseases and injuries that have been difficult to treat until now. Because regenerative medicine requires a large number of cells, the development of efficient cell culture methods has been actively pursued. For example, Patent Document 1 reports a method for culturing stem cells, which includes treating stem cells in a culture medium with a ROCK inhibitor.

[0003] Stable cell culture and proliferation requires periodic medium changes, subcultures, and other procedures. These procedures require repeated, complicated processes, placing a heavy burden on the operator, and there are also problems with the operator's skill affecting cell proliferation efficiency. For this reason, in recent years, cell culture devices (sometimes referred to herein as "bioreactors") that monitor the culture environment, such as medium pH and gas concentration, and automatically optimize these as necessary, have been developed and utilized. One example of such a bioreactor is the "ambr (registered trademark)" cell culture device from Sartorius. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2008-099662 Summary of the Invention [Problem to be solved by the invention]

[0005] The use of bioreactors can significantly reduce the burden on workers and problems associated with worker-related cell culture. However, at present, it is difficult to say that sufficient research has been done on the conditions that can optimize the efficiency of cell growth when using bioreactors.

[0006] Therefore, an object of the present invention is to provide a method for increasing the efficiency of cell growth in cell culture using a bioreactor by a simple and inexpensive means. [Means for solving the problem]

[0007] As a result of intensive research into the above-mentioned problems, the inventors discovered that highly desirable high-density culture can be achieved by preparing a large number of relatively small cell aggregates by controlling the cell aggregate size of the cells to be cultured. Based on this finding, they continued their research and completed the present invention. That is, the present invention is as follows.

[0008] [1] A method for culturing cells, comprising the following steps: A first step of preparing a population of cell clusters having a major axis of 400 μm or less; The second step is to culture the cell mass obtained in the first step in suspension. [2] The method described in [1], wherein the average major axis of the cell aggregates in the first step is 110 μm or less. [3] The method according to [1] or [2], wherein the median major axis of the cell aggregates in the first step is 90 μm or less. [4] The method according to any one of [1] to [3], wherein the first step and the second step are carried out continuously. [5] The method according to any one of [1] to [4], wherein the preparation of the cell cluster population in the first step is carried out using a spinner flask. [6] The method according to any one of [1] to [5], wherein the concentration of calcium ions in the medium used in the second step is 0.07 to 0.25 mM. [7] The method according to any one of [1] to [3], characterized in that the preparation of the cell cluster population in the first step is carried out by adjusting the calcium ion concentration, and the calcium ion concentration is 0.07 to 0.25 mM. [8] The method according to [6] or [7], further characterized in that the magnesium ion concentration is set to 0.5 to 0.65 mM. [9] The method according to any one of [1] to [8], wherein the suspension culture in the second step is carried out using a bioreactor.

[10] The method according to any one of [1] to [9], wherein the cells are stem cells.

[11] The method described in

[10] , wherein the stem cells are iPS cells.

[12] A method for culturing cells, comprising the step of culturing cells in suspension in a medium having a calcium ion concentration of 0.07 to 0.25 mM.

[13] The method described in

[12] , wherein the cells are stem cells.

[14] The method described in

[13] , wherein the stem cells are iPS cells. [Effects of the Invention]

[0009] According to the present invention, a preferable high-density culture can be achieved by a very inexpensive and simple method. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the change in iPS cell number over time in an example of the present application. [Figure 2] FIG. 2 shows the size distribution of cell aggregates prepared using a spinner flask in an example of the present application (on day 3, before suspension culture in a bioreactor, n=4). [Figure 3] FIG. 3 shows the change in cell number over time when cell cluster size was controlled and when it was not controlled. [Figure 4] FIG. 4 shows the size distribution of cell aggregates (at day 3) when cell aggregate size control was performed and when it was not performed. [Figure 5]FIG. 5 shows the change in cell number when iPS cells were cultured under conditions 1 to 3. [Figure 6] FIG. 6 shows the size distribution of cell clusters when iPS cells were cultured under conditions 1 to 3. [Figure 7] FIG. 7 shows the effect of Ca 2+ and Mg 2+ concentrations in the medium on the proliferation of iPS cells. [Figure 8] FIG. 8 shows the effect of Ca 2+ concentration in the medium on the size of cell clusters of iPS cells. [Figure 9] FIG. 9 shows the effect of Ca 2+ concentration in the medium on the viable cell density (VCD) of iPS cells. [Figure 10] FIG. 10 shows the effect of Ca 2+ concentration in the medium on the number of cell clusters of iPS cells. [Figure 11] FIG. 11 shows the effect of Ca 2+ concentration in the medium on the size of cell clusters of iPS cells. [Figure 12] FIG. 12 shows the effect of Ca 2+ concentration in the medium on the viable cell density (VCD) of iPS cells. [Figure 13] FIG. 13 shows the effect of Ca 2+ concentration in the medium on the number of cell clusters of iPS cells. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will be described in detail below.

[0012] definition As used herein, "suspension culture" refers to a cell culture method in which cells are not attached to a culture vessel. In the present invention, suspension culture may or may not involve external pressure or vibration applied to the liquid medium, or shaking or rotation in the liquid medium.

[0013] As used herein, the term "cell cluster" refers to an aggregate of cells formed by adhesion of cells when cells are subjected to suspension culture. Cell clusters are also sometimes called spheres (or spheroids).

[0014] In this specification, the size of cell clusters is determined by measuring their major axis. The size of cell clusters can be measured using a method known per se. The size of cell clusters can be easily measured using a commercially available device such as BZ-X710 (Keyence Corporation).

[0015] Cell culture method 1 The present invention provides a method for culturing cells (hereinafter, sometimes referred to as "Method 1 of the present invention"), comprising the following steps: A first step of preparing a population of cell clusters having a major axis of 400 μm or less; The second step is to culture the cell mass obtained in the first step in suspension.

[0016] The first step in Method 1 of the present invention aims to prepare a population of cell clusters of relatively small size.

[0017] In Method 1 of the present invention, the upper limit for the size of the cell clusters prepared in Step 1 (i.e., the longest diameter of the cell clusters) is 400 μm. Cell clusters with a longest diameter larger than this are undesirable from the standpoint of cell proliferation, as when subjected to further suspension culture, the cells located in the center of the cell clusters are unable to take up necessary components from the medium and are prone to apoptosis. Furthermore, when the cells are stem cells, not only are the cells in the center more prone to apoptosis, but they are also unable to maintain an undifferentiated state, raising concerns that this could result in a stem cell population of inconsistent quality.

[0018] In one embodiment of Method 1 of the present invention, the upper limit of the major axis of the cell mass prepared in the first step can be typically 400 μm, preferably 390 μm, 380 μm, 370 μm, 360 μm, 350 μm, 340 μm, 330 μm, 320 μm, 310 μm, 300 μm, 290 μm, 280 μm, 270 μm, 260 μm, 250 μm, 240 μm, 230 μm, 220 μm, 210 μm, or 200 μm.

[0019] Furthermore, in one embodiment of Method 1 of the present invention, the average long diameter of the cell aggregates is 400 μm or less, and preferably 300 μm or less, 250 μm or less, 200 μm or less, 190 μm or less, 180 μm or less, 170 μm or less, 160 μm or less, 150 μm or less, 140 μm or less, 130 μm or less, 120 μm or less, 110 μm or less, 100 μm or less, 95 μm or less, 90 μm or less, 85 μm or less, or 80 μm or less. In one aspect, the average major axis of the cell clusters may be 80 to 400 μm, 80 to 300 μm, 80 to 250 μm, 80 to 200 μm, 80 to 190 μm, 80 to 180 μm, 80 to 170 μm, 80 to 160 μm, 80 to 150 μm, 80 to 140 μm, 80 to 130 μm, 80 to 120 μm, 80 to 110 μm, 80 to 100 μm, 80 to 95 μm, 80 to 90 μm, or 80 to 85 μm.

[0020] In another embodiment of Method 1 of the present invention, the median major axis of the cell aggregates is 400 μm or less, and may preferably be 300 μm or less, 250 μm or less, 200 μm or less, 190 μm or less, 180 μm or less, 170 μm or less, 160 μm or less, 150 μm or less, 140 μm or less, 130 μm or less, 120 μm or less, 110 μm or less, 100 μm or less, 95 μm or less, 90 μm or less, 85 μm or less, 80 μm or less, 75 μm or less, or 70 μm or less. In one aspect, the median major axis of the cell clusters may be 70 to 400 μm, 70 to 300 μm, 70 to 250 μm, 70 to 200 μm, 70 to 190 μm, 70 to 180 μm, 70 to 170 μm, 70 to 160 μm, 70 to 150 μm, 70 to 140 μm, 70 to 130 μm, 70 to 120 μm, 70 to 110 μm, 70 to 100 μm, 70 to 95 μm, 70 to 90 μm, 70 to 85 μm, 70 to 80 μm, or 70 to 75 μm.

[0021] The first step of Method 1 of the present invention may be performed using any known method as long as it allows for the adjustment of the size of the cell clusters. For example, the first step of Method 1 of the present invention can be achieved by suspension culturing cells using a spinner flask. Briefly, when a certain number of cells are suspension cultured in a spinner flask, it is known that the lower the rotation speed of the blades, the larger the size of the cell clusters formed after a certain period of time, and the higher the rotation speed, the smaller the size of the cell clusters formed after a certain period of time. Therefore, those skilled in the art can easily prepare cell clusters of the desired size by appropriately determining the rotation speed of the blades of the spinner flask.

[0022] The spinner flask is not particularly limited as long as it can produce cell aggregates of the desired size, and commercially available spinner flasks can be used. When iPS cells are used as the cells, for example, a 30 mL single-use bioreactor for iPS cells (manufactured by ABLE, model number BWV-S03A) is preferably used.

[0023] Alternatively, a population of cell clusters having the above-mentioned preferred size may be prepared by first preparing a population of cell clusters of a relatively large size, and then dividing the cell clusters by passing them through a mesh or the like with an appropriate pore size.

[0024] In yet another embodiment, cell aggregates of a desired size can be prepared by suspension culture using low-attachment plates. Examples of low-attachment plates used in this method include, but are not limited to, Corning® Elplasia® plates.

[0025] The mesh is not particularly limited as long as it can be sterilized, and examples include nylon mesh and mesh made of metal such as stainless steel. The pore size of the mesh, for example, in the case of nylon mesh, is about 20 to 100 μm, preferably about 30 to 70 μm, and more preferably about 40 to 60 μm, but is not limited thereto. The shape of the mesh is also not particularly limited, but it is preferable that the mesh has a thickness and shape that minimizes damage to cells. For example, metal mesh such as stainless steel is preferred because it is easy to make the mesh thinner and causes relatively little damage to cells during division.

[0026] In another embodiment, the preparation of cell clusters having a desired size is carried out by adding calcium ions (Ca 2+ In another embodiment, the preparation of cell clusters having a desired size may be carried out by adjusting the concentration of calcium ions (Ca 2+ ) and magnesium ions (Mg 2+ This may be achieved by adjusting the concentration of ) in the medium.

[0027] The calcium ion concentration of the medium used in the step of preparing cell clusters of a relatively small size (i.e., the first step) is not particularly limited as long as cell clusters of the desired size can be prepared, but can be, for example, within the following concentration range: (1) Ca 2+ =0.07~0.25mM (2) Ca 2+ =0.10~0.25mM (3) Ca 2+ =0.13~0.25mM (4) Ca 2+ =0.18~0.25mM (5) Ca 2+ =0.20~0.25mM.

[0028] Furthermore, when adjusting the concentrations of calcium ions and magnesium ions, the concentration ranges of calcium ions and magnesium ions are not particularly limited as long as cell aggregates of the desired size can be prepared, but can be, for example, within the following concentration ranges: (6) Ca 2+ / Mg 2+ =0.07~0.25mM / 0.5~0.65mM (7) Ca 2+ / Mg 2+ =0.10~0.25mM / 0.5~0.65mM (8) Ca 2+ / Mg 2+ =0.13~0.25mM / 0.5~0.65mM (9) Ca 2+ / Mg 2+ =0.18~0.25mM / 0.5~0.65mM (10) Ca 2+ / Mg 2+ =0.20~0.25mM / 0.5~0.65mM.

[0029] The embodiment of the first step is not limited to the above two forms, and a method of dividing the cell mass using an enzyme instead of a mesh may also be used.

[0030] In one embodiment of Method 1 of the present invention, the preparation of the cell cluster population in Step 1 is carried out using a spinner flask. The use of a spinner flask is preferable because it is inexpensive and simple, and can minimize physical / chemical damage to the cell clusters.

[0031] The second step of Method 1 of the present invention aims to further grow the cells by subjecting the population of cell aggregates obtained in the first step to suspension culture.

[0032] The suspension culture in the second step is not particularly limited as long as the cells constituting the cell mass can grow, and any suspension culture method known per se may be used. In one aspect, suspension culture can be performed under known conditions using a commercially available bioreactor or the like.

[0033] In one embodiment, it is preferable to adjust the calcium ion concentration of the medium used in the second step of the method 1 of the present invention. The calcium ion concentration range of the medium used in the second step can be, for example, the following concentration range: (1) Ca 2+ =0.07~0.25mM (2) Ca 2+ =0.10~0.25mM (3) Ca 2+ =0.13~0.25mM (4) Ca 2+ =0.18~0.25mM (5) Ca 2+ =0.20~0.25mM.

[0034] In another embodiment, the calcium ion concentration range of the medium used in the second step of the method 1 of the present invention can be, for example, the following concentration range: (6) Ca 2+ =0.07~0.25mM (7) Ca 2+ =0.07~0.22mM (8) Ca 2+ =0.07~0.20mM (9) Ca 2+ =0.07~0.18mM (10) Ca 2+ =0.07~0.15mM.

[0035] As in step 1, the concentrations of calcium ions and magnesium ions in the medium used in step 2 may be adjusted. The preferred concentration ranges of calcium ions and magnesium ions in step 2 are not particularly limited as long as the desired effects of the present invention are obtained, but can be, for example, the following concentration ranges: (1) Ca 2+ / Mg 2+ =0.07~0.25mM / 0.5~0.65mM (2) Ca 2+ / Mg 2+ =0.10~0.25mM / 0.5~0.65mM (3) Ca 2+ / Mg 2+ =0.13~0.25mM / 0.5~0.65mM (4) Ca 2+ / Mg 2+ =0.18~0.25mM / 0.5~0.65mM (5) Ca 2+ / Mg 2+ =0.20~0.25mM / 0.5~0.65mM.

[0036] In one embodiment of Method 1 of the present invention, it is also preferable to set the calcium ion and / or magnesium ion concentrations of the medium used in Step 1 to the calcium and / or magnesium ion concentrations commonly used in medium (e.g., calcium ion: 0.8 to 1.2 mM, magnesium ion: 0.8 to 1.2 mM) without reducing them to the above-mentioned ranges, and to set only the calcium ion and / or magnesium ion concentrations of the medium used in Step 2 to the above-mentioned concentration ranges.

[0037] In another embodiment of Method 1 of the present invention, Step 1 and Step 2 can also be carried out consecutively. More specifically, if a desired high-density culture can be achieved by preparing a large number of relatively small cell aggregates in a certain culture system and then continuing the culture without changing the culture system, there is no need to clearly set Step 2, and the subsequent performance of Step 1 can be replaced with the performance of Step 2.

[0038] The cell types to which Method 1 of the present invention can be applied are not particularly limited as long as they form spheres when subjected to suspension culture. Examples of such cell types include germ cells such as sperm and eggs, somatic cells that constitute a living organism, stem cells (e.g., pluripotent stem cells), progenitor cells, cancer cells isolated from a living organism, cells isolated from a living organism that have acquired immortalization and are stably maintained ex vivo (cell lines), cells isolated from a living organism that have been artificially genetically modified, and cells isolated from a living organism that have undergone artificial nucleus exchange. Examples of somatic cells that make up a living organism include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, osteocytes, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g., smooth muscle cells or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells (e.g., CD34-positive cells derived from umbilical cord blood), and mononuclear cells. Such somatic cells include cells obtained from any tissue, such as skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood (including umbilical cord blood), bone marrow, heart, eye, brain, or neural tissue.

[0039] Stem cells are cells that have the ability to replicate themselves and differentiate into cells of multiple lineages. Examples include, but are not limited to, embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, and hair follicle stem cells.

[0040] A cell line is a cell that has acquired the ability to proliferate indefinitely through artificial manipulation outside of a living body. Examples of cell lines include, but are not limited to, CHO (Chinese hamster ovary cell line), HCT116, Huh7, HEK293 (human embryonic kidney cells), HeLa (human uterine cancer cell line), HepG2 (human liver cancer cell line), UT7 / TPO (human leukemia cell line), MDCK, MDBK, BHK, C-33A, HT-29, AE-1, 3D9, Ns0 / 1, Jurkat, NIH3T3, PC12, S2, Sf9, Sf21, High Five (registered trademark), and Vero.

[0041] In a preferred embodiment of Method 1 of the present invention, the cells are stem cells, more preferably iPS cells.

[0042] The medium used in Step 1 and Step 2 of Method 1 of the present invention is not particularly limited as long as it allows cell growth. The medium used in Step 1 and Step 2 may be the same or different. The medium used may be prepared by a method known per se depending on the cells to be cultured, or may be a commercially available product.

[0043] Examples of media that can be used include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A Medium, Minimum Essential Medium (MEM), Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle's Minimum Essential Medium (αMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, and Fischer's Medium.

[0044] Furthermore, media particularly used for culturing stem cells include STEMPRO (registered trademark) hESC SFM medium (Life Technologies), mTeSR1 medium (STEMCELL Technologies), TeSR2 medium (STEMCELL Technologies), TeSR-E8 medium (STEMCELL Technologies), Essential 8 medium (Life Technologies), HEScGRO (trademark) Serum-Free Medium for hES cells (Millipore), PluriSTEM (trademark) Human ES / iPS Medium (EMD Millipore), NutriStem (registered trademark) hESC XF medium (Biological Industries Israel Beit-Haemek), NutriStem (trademark) XF / FF Culture Medium (Stemgent), AF NutriStem (registered trademark) hESC XF medium (Biological Industries Israel Beit-Haemek), S-medium (DS Pharma Biomedical Co., Ltd.), and StemFit (registered trademark). Examples include AK03N medium (Ajinomoto Co., Inc.), hESF9 medium, hESF-FX medium, CDM medium, DEF-CS 500 Xeno-Free 3D Spheroid Culture Medium (Cellartis), and StemFlex medium (Thermo Fisher Scientific).

[0045] In one embodiment of Method 1 of the present invention, components favorable for cell growth can be further added to the medium used in Step 1 and / or Step 2. Examples of such components include sugars such as glucose, fructose, sucrose, and maltose; amino acids such as asparagine, aspartic acid, glutamine, and glutamic acid; proteins such as albumin and transferrin; peptides such as glycylglycylglycine and soybean peptide; serum; vitamins such as choline, vitamin A, B vitamins (thiamine, riboflavin, pyridoxine, cyanocobalamin, biotin, folic acid, pantothenic acid, and nicotinamide), vitamin C, and vitamin E; fatty acids such as oleic acid, arachidonic acid, and linoleic acid; lipids such as cholesterol; inorganic salts such as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, and sodium dihydrogen phosphate; trace elements such as zinc, copper, and selenium; and N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid). buffers such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (BES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and N-[tris(hydroxymethyl)methyl]glycine (Tricine); antibiotics such as amphotericin B, kanamycin, gentamicin, streptomycin, and penicillin; Type I collagen, Type II collagen Examples of suitable components include cell adhesion factors and extracellular matrix components such as collagen, fibronectin, laminin, poly-L-lysine, and poly-D-lysine; cytokines and growth factors such as interleukin, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), transforming growth factor (TGF)-α, transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), and activin A; and hormones such as dexamethasone, hydrocortisone, estradiol, progesterone, glucagon, and insulin. Appropriate components can be selected and used depending on the type of cells to be cultured.

[0046] In a preferred embodiment of Method 1 of the present invention, the medium used in Step 1 and / or Step 2 may additionally contain D-glucose and five amino acids (tryptophan, serine, cysteine ​​(or cystine), methionine, and arginine).

[0047] Glucose (or a salt thereof) can be added to the medium so that the glucose concentration is usually 0.1 g / L / day to 900 g / L / day, preferably 1 g / L / day to 200 g / L / day, and more preferably 1 g / L / day to 20 g / L / day.

[0048] Furthermore, the five amino acids (tryptophan, serine, cysteine ​​(cystine), methionine, and arginine) are adjusted relative to the medium so that the tryptophan concentration (concentration converted to free tryptophan) is usually 0.1 mg / L / day to 11,000 mg / L / day, preferably 1 mg / L / day to 1,000 mg / L / day, and more preferably 1 mg / L / day to 100 mg / L / day, the serine concentration (concentration converted to free serine) is usually 0.1 mg / L / day to 425,000 mg / L / day, preferably 1 mg / L / day to 1,000 mg / L / day, and more preferably 1 mg / L / day to 100 mg / L / day, and the cysteine ​​or cystine concentration (concentration converted to free cysteine) is usually 0. It can be added so that the concentration of methionine (concentration converted to free methionine) is usually 0.1 mg / L / day to 55000 mg / L / day, preferably 1 mg / L / day to 1000 mg / L / day, more preferably 1 mg / L / day to 100 mg / L / day, and the concentration of arginine (concentration converted to free arginine) is usually 0.1 mg / L / day to 150000 mg / L / day, preferably 1 mg / L / day to 2000 mg / L / day, more preferably 1 mg / L / day to 200 mg / L / day.

[0049] When Method 1 of the present invention is applied to stem cells, in a preferred embodiment, bFGF may be additionally added at regular intervals to the medium used in the first step and / or the second step.

[0050] In Method 1 of the present invention, cell culture conditions may be selected from known methods depending on the cell type. For example, the culture temperature may be typically 25°C to 39°C, preferably 33°C to 39°C. The carbon dioxide concentration may be typically 4% to 10% by volume, preferably 4% to 6% by volume. The oxygen concentration may be typically 1% to 25% by volume, preferably 4% to 20% by volume. The frequency of medium replacement may be once every 2 to 3 days, once a day, or multiple times a day (e.g., twice a day). The medium may be replaced in its entirety or partially (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%). The amount of medium replaced may be determined appropriately depending on the cell type, etc.

[0051] Cell culture method 2 The present invention also provides a cell culture method (hereinafter sometimes referred to as "Method 2 of the present invention"), which comprises a step of suspension culturing cells in a medium having a calcium ion concentration of 0.07 to 0.25 mM.

[0052] The cell types to which Method 2 of the present invention can be applied are not particularly limited as long as they form spheres when subjected to suspension culture. Examples of such cell types include germ cells such as sperm and eggs, somatic cells that constitute a living organism, stem cells (e.g., pluripotent stem cells), progenitor cells, cancer cells isolated from a living organism, cells isolated from a living organism that have acquired immortalization and are stably maintained ex vivo (cell lines), cells isolated from a living organism that have been artificially genetically modified, and cells isolated from a living organism that have undergone artificial nucleus exchange. Examples of somatic cells that make up a living organism include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, osteocytes, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g., smooth muscle cells or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells (e.g., CD34-positive cells derived from umbilical cord blood), and mononuclear cells. Such somatic cells include cells obtained from any tissue, such as skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood (including umbilical cord blood), bone marrow, heart, eye, brain, or neural tissue.

[0053] Stem cells are cells that have the ability to replicate themselves and differentiate into cells of multiple lineages. Examples include, but are not limited to, embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, and hair follicle stem cells.

[0054] A cell line is a cell that has acquired the ability to proliferate indefinitely through artificial manipulation outside of a body. Examples of cell lines include, but are not limited to, CHO (Chinese hamster ovary cell line), HCT116, Huh7, HEK293 (human embryonic kidney cells), HeLa (human uterine cancer cell line), HepG2 (human liver cancer cell line), UT7 / TPO (human leukemia cell line), MDCK, MDBK, BHK, C-33A, HT-29, AE-1, 3D9, Ns0 / 1, Jurkat, NIH3T3, PC12, S2, Sf9, Sf21, High Five (registered trademark), and Vero.

[0055] In a preferred embodiment of Method 2 of the present invention, the cells are stem cells, more preferably iPS cells.

[0056] Examples of media that can be used in Method 2 of the present invention include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Minimum Essential Medium (MEM), Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle's Minimum Essential Medium (αMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, and Fischer's Medium.

[0057] Furthermore, media particularly used for culturing stem cells include STEMPRO (registered trademark) hESC SFM medium (Life Technologies), mTeSR1 medium (STEMCELL Technologies), TeSR2 medium (STEMCELL Technologies), TeSR-E8 medium (STEMCELL Technologies), Essential 8 medium (Life Technologies), HEScGRO (trademark) Serum-Free Medium for hES cells (Millipore), PluriSTEM (trademark) Human ES / iPS Medium (EMD Millipore), NutriStem (registered trademark) hESC XF medium (Biological Industries Israel Beit-Haemek), NutriStem (trademark) XF / FF Culture Medium (Stemgent), AF NutriStem (registered trademark) hESC XF medium (Biological Industries Israel Beit-Haemek), S-medium (DS Pharma Biomedical Co., Ltd.), and StemFit (registered trademark). Examples include AK03N medium (Ajinomoto Co., Inc.), hESF9 medium, hESF-FX medium, CDM medium, DEF-CS 500 Xeno-Free 3D Spheroid Culture Medium (Cellartis), and StemFlex medium (Thermo Fisher Scientific).

[0058] In one embodiment of Method 2 of the present invention, the medium used can further contain components favorable for cell growth. Examples of such components include sugars such as glucose, fructose, sucrose, and maltose; amino acids such as asparagine, aspartic acid, glutamine, and glutamic acid; proteins such as albumin and transferrin; peptides such as glycylglycylglycine and soybean peptide; serum; vitamins such as choline, vitamin A, B vitamins (thiamine, riboflavin, pyridoxine, cyanocobalamin, biotin, folic acid, pantothenic acid, and nicotinamide), vitamin C, and vitamin E; fatty acids such as oleic acid, arachidonic acid, and linoleic acid; lipids such as cholesterol; inorganic salts such as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, and sodium dihydrogen phosphate; trace elements such as zinc, copper, and selenium; and N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid). buffers such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (BES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and N-[tris(hydroxymethyl)methyl]glycine (Tricine); antibiotics such as amphotericin B, kanamycin, gentamicin, streptomycin, and penicillin; Type I collagen, Type II collagen Examples of suitable components include cell adhesion factors and extracellular matrix components such as collagen, fibronectin, laminin, poly-L-lysine, and poly-D-lysine; cytokines and growth factors such as interleukin, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), transforming growth factor (TGF)-α, transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), and activin A; and hormones such as dexamethasone, hydrocortisone, estradiol, progesterone, glucagon, and insulin. Appropriate components can be selected and used depending on the type of cells to be cultured.

[0059] Method 2 of the present invention is characterized by appropriately adjusting the calcium ion concentration in the medium. The calcium ion concentration in the medium used is generally, but not limited to, 0.07 to 0.25 mM, preferably 0.10 to 0.25 mM, more preferably 0.13 to 0.25 mM, even more preferably 0.18 to 0.25 mM, and particularly preferably 0.20 to 0.25 mM.

[0060] In another embodiment, in Method 2 of the present invention, the range of calcium ion concentration contained in the medium is usually 0.07 to 0.25 mM, preferably 0.07 to 0.22 mM, more preferably 0.07 to 0.20 mM, even more preferably 0.07 to 0.18 mM, and particularly preferably 0.07 to 0.15 mM, but is not limited to these.

[0061] In one embodiment, the calcium ion concentration and magnesium ion concentration may be adjusted. The range of calcium ion and magnesium ion concentrations contained in the medium is not particularly limited as long as the desired effect is obtained, but can be set as follows: (1) Ca 2+ / Mg 2+ =0.07~0.25mM / 0.5~0.65mM (2) Ca 2+ / Mg 2+ =0.10~0.25mM / 0.5~0.65mM (3) Ca 2+ / Mg 2+ =0.13~0.25mM / 0.5~0.65mM (4) Ca 2+ / Mg 2+ =0.18~0.25mM / 0.5~0.65mM (5) Ca 2+ / Mg 2+ =0.20~0.25mM / 0.5~0.65mM.

[0062] In another embodiment, the ranges of calcium ion and magnesium ion concentrations contained in the medium are not particularly limited as long as the desired effect is obtained, but can be set as follows: (6) Ca2+ / Mg 2+ =0.07~0.25mM / 0.5~0.65mM (7) Ca 2+ / Mg 2+ =0.07~0.22mM / 0.5~0.65mM (8) Ca 2+ / Mg 2+ =0.07~0.20mM / 0.5~0.65mM (9) Ca 2+ / Mg 2+ =0.07~0.18mM / 0.5~0.65mM (10) Ca 2+ / Mg 2+ =0.07~0.15mM / 0.5~0.65mM.

[0063] In a preferred embodiment of Method 2 of the present invention, the medium used may additionally contain D-glucose and five amino acids (tryptophan, serine, cysteine ​​(or cystine), methionine, and arginine).

[0064] Glucose (or a salt thereof) can be added to the medium so that the glucose concentration is usually 0.1 g / L / day to 900 g / L / day, preferably 1 g / L / day to 200 g / L / day, and more preferably 1 g / L / day to 20 g / L / day.

[0065] Furthermore, the five amino acids (tryptophan, serine, cysteine ​​(cystine), methionine, and arginine) are adjusted relative to the medium so that the tryptophan concentration (concentration converted to free tryptophan) is usually 0.1 mg / L / day to 11,000 mg / L / day, preferably 1 mg / L / day to 1,000 mg / L / day, and more preferably 1 mg / L / day to 100 mg / L / day, the serine concentration (concentration converted to free serine) is usually 0.1 mg / L / day to 425,000 mg / L / day, preferably 1 mg / L / day to 1,000 mg / L / day, and more preferably 1 mg / L / day to 100 mg / L / day, and the cysteine ​​or cystine concentration (concentration converted to free cysteine) is usually 0. It can be added so that the concentration of methionine (concentration converted to free methionine) is usually 0.1 mg / L / day to 55000 mg / L / day, preferably 1 mg / L / day to 1000 mg / L / day, more preferably 1 mg / L / day to 100 mg / L / day, and the concentration of arginine (concentration converted to free arginine) is usually 0.1 mg / L / day to 150000 mg / L / day, preferably 1 mg / L / day to 2000 mg / L / day, more preferably 1 mg / L / day to 200 mg / L / day.

[0066] When Method 2 of the present invention is applied to stem cells, in a preferred embodiment, bFGF may be additionally added to the culture medium used at regular intervals.

[0067] In Method 2 of the present invention, cell culture conditions may be selected from known methods depending on the cell type. For example, the culture temperature may be typically 25°C to 39°C, preferably 33°C to 39°C. The carbon dioxide concentration may be typically 4% to 10% by volume, preferably 4% to 6% by volume. The oxygen concentration may be typically 1% to 25% by volume, preferably 4% to 20% by volume. The frequency of medium replacement may be once every 2 to 3 days, once a day, or multiple times a day (e.g., twice a day). The medium may be replaced in its entirety or partially (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%). The amount of medium replaced may be determined appropriately depending on the cell type, etc.

[0068] In the method 2 of the present invention, the seeding concentration of cells at the start of culture is usually 1 × 10 4 cells / mL ~ 1 × 10 7 cells / mL, preferably 5 x 10 4 cells / mL ~ 5 × 10 6 cells / mL, preferably 5×10 4 cells / mL ~ 1 × 10 6 cells / mL, particularly preferably 1 × 10 5 cells / mL ~ 1 × 10 6 The concentration may be, but is not limited to, cells / mL.

[0069] The present invention will be described in more detail in the following examples, but the present invention is not limited to these examples. In this specification and examples, particle size is measured by number. [Example]

[0070] [Example 1] Sphere formation and cell proliferation of iPS cells using spinner flasks Using a 30 mL single-use bioreactor for iPS cells (ABLE, model number BWV-S03A), 6 × 10 iPS cell line 1231A3 was cultured in StemFit® AK03N + 10 μM Y-27632 (Wako, 034-24024). 5Cells were seeded at a density of 1000 cells / mL and cultured in a CO2 incubator at 37°C, 5% CO2, and 120 rpm. On day 2, 70% of the medium was replaced with StemFit AK03N. On day 3, 10 mL of the cell suspension was resuspended in fresh StemFit AK03N + 200 ng / mL bFGF (Peprotech) and transferred to an ambr15 bioreactor (Sartorius, model number 001-0881) for culture at 37°C, pH 7.2, 4% dissolved oxygen, and 300 rpm. The length of the cell clusters was quantified using a BZ-X710 (Keyence) microscope (the cutoff value was set to 50 μm to measure the length of the cell clusters only). Seventy percent of the medium was replaced twice daily with StemFit AK03N + 200 ng / mL bFGF, and the medium was further supplemented with 40 mg / L / day Trp (Ajinomoto Co.), 40 mg / L / day Ser (Ajinomoto Co.), 40 mg / L / day Cys hydrochloride (Nippon Protein Co.), 40 mg / L / day Met (Ajinomoto Co.), 160 mg / L / day Arg (Ajinomoto Co.), and 4 g / L / day D-glucose (Nacalai Tesque, model number 16806-25). After day 5 of culture, viable cell counts were measured using the Vi-CELL viability analyzer. TM Measurement was performed using a Beckman Coulter XR.

[0071] The results of a single test of iPS cell proliferation are shown in Figure 1. The results of a quadruple test of the distribution of the long diameter of the cell clusters when transferred to the bioreactor on day 3 are shown in Figure 2 and Table 1 below. By controlling the long diameter of the cell clusters to a size of 400 μm or less, the distribution of the long diameter of the cell clusters was reduced to 1.0 × 10 7 It was shown that high-density culture exceeding cells / mL can be achieved.

[0072] [Table 1]

[0073] [Example 2] Effect of sphere size control on proliferation To confirm that sphere size control is important for cell proliferation, we cultured iPS cells in a microbioreactor without controlling sphere size. Specifically, 6 × 10 iPS cells from the 1210B2 strain were cultured in the ambr15 microbioreactor. 5 Cells were seeded at a cell density of 1000 cells / mL and cultured in a CO2 incubator using StemFit AK03N + 10uM Y-27632 at 37°C, CO2 concentration = 5%, and agitation speed = 300 rpm. On the second day of seeding, 70% of the medium was replaced with StemFit AK03N. On the third day of seeding, 10mL of the cell suspension was resuspended in fresh StemFit AK03N, and then 70% of the medium was replaced twice daily with StemFit AK03N. Furthermore, 40mg / L / day Trp, 40mg / L / day Ser, 40mg / L / day Cys, 40mg / L / day Met, 160mg / L / day Arg, and 4g / L / day D-glucose were added. Meanwhile, the sphere size control group was treated in the same manner as in Example 1 using StemFit AK03N. The major axis of the spheres was quantified using BZ-X710 on day 3 of culture. In addition, the number of viable cells was measured using a viable cell autoanalyzer, Vi-CELL (trademark) XR, on day 7 of culture or later.

[0074] Figure 3 shows the results of a series of experiments examining the proliferation of iPS cells. Figure 4 also shows the results of a series of experiments examining the distribution of the long diameter of the spheres when they were transferred to the microbioreactor on day 3. Even under similar culture conditions, if the sphere size is not controlled, the number of cells will be 1.0 x 10 7 It was shown that the number of cells / mL was not reached.

[0075] [Example 3] Examination of sphere size control method In the following experimental example, we investigated components that can maintain small sphere size when growing induced pluripotent stem cells (iPS cells) in suspension culture. The iPS cells used were the 1231A3 strain purchased from iPS Academia Japan. The commercially available iPS cell medium, StemFit AK03N (Ajinomoto Co.), was used.

[0076] Using a 30 mL single-use bioreactor for iPS cells (ABLE: BWV-S03A), the concentrations of calcium chloride, magnesium sulfate, and magnesium chloride were adjusted to the three conditions shown in Table 2. 2 × 10 1231A3 iPS cells were added to StemFit AK03N + 10 μM Y-27632 (Wako: 034-24024). 5 The cells were seeded at a cell density of 1000 cells / mL and cultured in a CO2 incubator at 37°C, CO2 concentration of 5%, and agitation speed of 55 rpm. From the second day after seeding, 70% of the medium was replaced daily with StemFit AK03N under each condition. On the seventh day of culture, the viable cell count was measured using the Vi-CELL viability cell autoanalyzer. TM The diameter of the cell clusters was measured using a BZ-X710 (Keyence).

[0077] [Table 2]

[0078] The results of a single test of iPS cell proliferation are shown in Figure 5. The results of a single test of the distribution of the major diameter of spheres on day 7 are shown in Figure 6 and Table 3. 2+ and Mg 2+ It was shown that the sphere length can be kept small by adjusting the concentration of

[0079] [Table 3]

[0080] [Example 4] Examination of the effect of magnesium ion and calcium ion concentrations on cell proliferation Using a 30 mL single-use bioreactor for iPS cells (ABLE: BWV-S03A), 6 × 10 iPS cell line 1210B2 cells were cultured in StemFit AK03N + 10 μM Y-27632 (Wako: 034-24024). 5Cells were seeded at a cell density of 1000 cells / mL and cultured in a CO2 incubator at 37°C, 5% CO2, and 120 rpm. On the second day after seeding, 70% of the medium was replaced with StemFit AK03N. On the third day after seeding, 10 mL of the cell suspension was resuspended in fresh StemFit AK03N medium containing the calcium chloride, magnesium sulfate, and magnesium chloride adjusted to the two conditions shown in Table 4. The medium was then transferred to a microbioreactor, ambr15 (sartorius: 001-0881), and cultured at 37°C, pH 7.2, 20% dissolved oxygen, and 300 rpm. Seventy percent of the medium was replaced twice daily with StemFit AK03N for each condition. Furthermore, both groups were supplemented with 40 mg / L / day Trp (Ajinomoto Co.), 40 mg / L / day Ser (Ajinomoto Co.), 40 mg / L / day Cys hydrochloride (Nippon Protein Co.), 40 mg / L / day Met (Ajinomoto Co.), 160 mg / L / day Arg (Ajinomoto Co.), and 4 g / L / day D-glucose (Nacalai Tesque: 16806-25). On day 7 of culture, viable cell counts were measured using the Vi-CELL viability analyzer. TM Measurement was performed using a Beckman Coulter XR.

[0081] [Table 4]

[0082] Ca effects on iPS cell proliferation 2+ and Mg 2+ The results of the two-part test are shown in Figure 7. As shown in Figure 7, 2+ and Mg 2+ It was shown that at certain concentrations, it has a significant effect on cell proliferation.

[0083] [Example 5] Study of sphere size control method 2 In Example 3 above, Ca 2+ and Mg 2+ It was shown that the concentration of Ca is important for controlling the sphere length. 2+ and Mg 2+We investigated which of the concentrations has a greater effect on controlling the sphere length.

[0084] Using a 30 mL single-use bioreactor for iPS cells (ABLE: BWV-S03A), 6 × 10 iPS cell line 1210B2 cells were cultured in StemFit AK03N + 10 μM Y-27632 (Wako: 034-24024). 5 The cells were seeded at a cell density of 1000 cells / mL and cultured in a CO2 incubator at 37°C, CO2 concentration of 5%, and agitation speed of 120 rpm. On the second day after seeding, 70% of the medium was replaced with StemFit AK03N. (The calcium ion concentration of the medium used up to this point was not adjusted. Therefore, the calcium ion concentration of the medium was similar to that of general cell culture medium (approximately 0.8 mM to 1.2 mM).) On the third day after seeding, 10 mL of the cell suspension was resuspended in fresh StemFit AK03N prepared under the two conditions shown in Table 5, with the calcium chloride concentration adjusted. The cell suspension was transferred to a microbioreactor, ambr15 (sartorius: 001-0881), and cultured under agitation conditions of 37°C, pH 7.2, dissolved oxygen concentration of 20%, and agitation speed of 300 rpm. Note that under conditions 1 and 2, Mg 2+The concentrations of α- and β-actin were the same (1.003 mM). 70% of the medium was replaced twice daily with StemFit AK03N for each condition, and both groups were further supplemented with 40 mg / L / day Trp (Ajinomoto Co., Inc.), 40 mg / L / day Ser (Ajinomoto Co., Inc.), 40 mg / L / day Cys hydrochloride (Nippon Protein Co., Ltd.), 40 mg / L / day Met (Ajinomoto Co., Inc.), 160 mg / L / day Arg (Ajinomoto Co., Inc.), 18.6 mg / L / day His (Ajinomoto Co., Inc.), 43.7 mg / L / day Ile (Ajinomoto Co., Inc.), 47.3 mg / L / day Leu (Ajinomoto Co., Inc.), 73.1 mg / L / day Lys hydrochloride (Ajinomoto Co., Inc.), 28.4 mg / L / day Phe (Ajinomoto Co., Inc.), 42.3 mg / L / day Val (Ajinomoto Co., Inc.), and 4 g / L / day D-glucose (Nacalai Tesque: 16806-25) was added. The major axis of the spheres was quantified using a BZ-X710 (Keyence) on days 3 and 6 of culture. The number of viable cells was also measured on days 3 and 6 of culture using a Vi-CELL™ XR (Beckman Coulter) viable cell autoanalyzer.

[0085] [Table 5]

[0086] Ca 2+ The results of a series of experiments examining the effect of concentration on iPS cell sphere size are shown in Figure 8 and Table 6. The VCD is shown in Figure 9, and the number of cell clusters is shown in Figure 10.

[0087] As is clear from these figures and tables, Ca 2+ It was shown that by adjusting the concentration, the sphere length can be kept small and the number of spheres can be kept large.

[0088] [Table 6]

[0089] [Example 6] Effect on cell proliferation of iPS cells using a suspension culture system 1.72 × 10 iPS cells 1210B2 strain were added to the microbioreactor ambr15 (sartorius:001-0881). 5 The cells were seeded at a cell density of 1000 cells / mL, and the calcium chloride concentration was adjusted. Fresh StemFit AK03N + 10 μM Y-27632 adjusted to the two conditions shown in Table 7 was used for agitation culture at 37°C, pH 7.2, dissolved oxygen concentration 20%, and agitation speed 300 rpm. From the second day of culture onwards, 70% of the medium was replaced twice daily with StemFit AK03N for each condition, and both groups were further supplemented with 40 mg / L / day Trp (Ajinomoto Co.), 40 mg / L / day Ser (Ajinomoto Co.), 40 mg / L / day Cys hydrochloride (Nippon Protein Co.), 40 mg / L / day Met (Ajinomoto Co.), 160 mg / L / day Arg (Ajinomoto Co.), 18.6 mg / L / day His (Ajinomoto Co.), 43.7 mg / L / day Ile (Ajinomoto Co.), 47.3 mg / L / day Leu (Ajinomoto Co.), 73.1 mg / L / day Lys hydrochloride (Ajinomoto Co.), 28.4 mg / L / day Phe (Ajinomoto Co.), 42.3 mg / L / day Val (Ajinomoto Co.), and 4 g / L / day D-glucose (Nacalai Tesque: 16806-25) was added. On day 6, the sphere length was quantified using a BZ-X710 (Keyence). Also on day 6 of culture, the number of viable cells was measured using a Vi-CELL™ XR (Beckman Coulter) viable cell autoanalyzer.

[0090] [Table 7]

[0091] Ca 2+ The results of a series of tests on the effect of concentration on the sphere size of iPS cells are shown in Figure 11 and Table 8. The VCD is shown in Figure 12, and the number of cell clusters in Figure 13. 2+ It was shown that by adjusting the concentration, it is possible to maintain a small sphere diameter and a large sphere number.

[0092] [Table 8] [Industrial Applicability]

[0093] According to the present invention, it is possible to achieve a more preferable high-density culture than in the previous embodiments, using a very inexpensive and simple method.

[0094] This application is based on patent application No. 2020-003961 (filing date: January 14, 2020) and patent application No. 2020-096485 (filing date: June 2, 2020) filed in Japan, the contents of which are incorporated in their entirety herein.

Claims

1. The following steps: A first step of preparing a population of cell clusters having a major axis of 400 μm or less; A second step of suspension culture of the cell aggregates obtained in the first step. A method for culturing cells, comprising: the concentration of calcium ions in the medium used in the second step is 0.07 to 0.25 mM; The method, wherein the cell is an iPS cell or an ES cell.

2. 2. The method according to claim 1, wherein the average major axis of the cell aggregates in the first step is 110 μm or less.

3. 3. The method according to claim 1, wherein the median length of the cell aggregates in the first step is 90 μm or less.

4. The method according to any one of claims 1 to 3, wherein the first and second steps are carried out continuously.

5. The method according to any one of claims 1 to 4, wherein the preparation of the cell cluster population in the first step is carried out using a spinner flask.

6. The method according to any one of claims 1 to 3, wherein the preparation of the cell cluster population in the first step is carried out by adjusting the concentration of calcium ions, and the concentration of calcium ions is 0.07 to 0.25 mM.

7. The method according to claim 1 or 6, characterized in that the concentration of magnesium ions in the first and / or second steps is 0.5 to 0.65 mM.

8. The method according to any one of claims 1 to 7, wherein the suspension culture in the second step is carried out using a bioreactor.

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