novel culture method

Abstract

In the method of culturing cells, the cells are cultured using a culture solution in which the ratio of the mass of calcium ions to the mass of sodium ions is adjusted to 2.8 mass% or more and 48.5 mass% or less.

Description

Technical Field

[0001] This disclosure relates to a new cultivation method. Background Technology

[0002] In fields such as drug production, gene therapy, regenerative medicine, and immunotherapy, there is a need for efficient and large-scale culture of cells and tissues. Microcarrier culture is a known technique for large-scale cell culture. Microcarrier culture involves, for example, introducing cells, culture medium, and microcarriers that serve as adhesion scaffolds into a culture vessel; intermittently stirring the culture medium to suspend the cells and microcarriers; and allowing the suspended cells to descend and contact the microcarriers, adhering to and proliferating on the surface of the microcarriers (see, for example, Patent Document 1). Microcarriers provide a very large surface area relative to their volume for adhesion and proliferation, which is advantageous for large-scale cell culture.

[0003] To date, cell culture carriers containing alginate and cell culture methods using such carriers are known (see, for example, Patent Documents 2 and 3). However, cell culture carriers containing calcium alginate have the following problems: they cannot maintain a suitable shape (e.g., spherical, near-spherical, etc.) due to culture conditions, some of them break down, resulting in reduced cell culture efficiency.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: International Publication No. 2010 / 138702

[0007] Patent Document 2: Japanese Patent Application Publication No. 2021-153441

[0008] Patent Document 3: Japanese Patent Application Publication No. 5-81 Summary of the Invention

[0009] The problem that the invention aims to solve

[0010] Under such circumstances, it is still necessary to seek more effective cell culture methods.

[0011] Methods for solving problems

[0012] The inventors have discovered that in a culture medium containing sodium and calcium ions, where the mass ratio (mass concentration) of calcium ions to sodium ions is 2.8% or more and 48.5% or less, using a cell culture carrier comprising a polysaccharide cross-linked with divalent or higher cations can stably maintain the shape of the cell culture microcarrier, resulting in high-efficiency cell culture. This disclosure is based on these insights.

[0013] That is, according to one aspect of this disclosure, a method for culturing cells is provided.

[0014] The method includes a culture step of culturing the cells in a culture medium using a cell culture carrier containing polysaccharides cross-linked by divalent or higher cations.

[0015] The ratio of calcium ion mass to sodium ion mass in the culture medium is greater than 2.8% by mass and less than 48.5% by mass.

[0016] Invention Effects

[0017] Since the culture medium disclosed herein can stably maintain the shape of the cell culture microcarrier, it is advantageous in cell culture, especially in cell culture using cell culture microcarriers, in terms of high-efficiency cell culture. Attached Figure Description

[0018] [ Figure 1 [Image] Represents a phase-contrast microscope image of a microcarrier used for cell culture.

[0019] [ Figure 2 [Image showing a phase-contrast microscope image of a dried microcarrier used for cell culture.]

[0020] [ Figure 3 This image shows a phase-contrast microscope image of a cell culture microcarrier in the culture medium on day 7 of culture.

[0021] [ Figure 4 This image shows a fluorescence microscope image of live cells stained with calcein-AM solution on day 7 of culture. Detailed Implementation

[0022] <Cultural medium for cell culture>

[0023] According to one aspect of this disclosure, a culture medium (also referred to herein as "the culture medium for cell culture of this disclosure", or simply "the culture medium of this disclosure", etc.) is provided, which is a culture medium for cell culture and contains sodium ions and calcium ions, wherein the mass ratio of the calcium ions to the mass of the sodium ions is 2.8% by mass or more and 48.5% by mass or less.

[0024] The culture medium disclosed herein can stably maintain the shape of the cell culture microcarriers. Therefore, in cell culture, especially in cell culture using cell culture carriers comprising polysaccharides cross-linked by divalent or higher cations, and particularly preferably in cell culture using cell culture microcarriers comprising polysaccharides cross-linked by divalent or higher cations, cells can be cultured with high efficiency. Therefore, the culture medium disclosed herein is preferably used in cell culture using cell culture carriers comprising polysaccharides cross-linked by divalent or higher cations, and particularly in cell culture microcarriers comprising polysaccharides cross-linked by divalent or higher cations.

[0025] In this disclosure, the "binding" of the components constituting the cell culture carrier to each other includes all types of binding, including both binding accompanied by chemical binding and binding without chemical binding. The binding of the components constituting the cell culture carrier to each other may include one type of binding or two or more different types of binding. Furthermore, examples of the aforementioned binding of the components constituting the cell culture carrier to each other include the binding of alginate to each other, the binding of alginate to a divalent or higher cation, the binding of alginate to other components (components other than alginate and divalent or higher cations), and the binding of other components to each other.

[0026] "Cross-linking" is a mode of "binding." In this disclosure, "cross-linked," "bound by cross-linking," and similar expressions are used interchangeably, all referring to the reversible or irreversible binding of multiple molecules, either through or without other substances. For example, "alginic acid cross-linked by divalent or higher cations" refers to the reversible or irreversible binding of multiple alginate molecules through divalent or higher cations. In this disclosure, unless otherwise defined, "chemical cross-linking" and similar expressions refer to the irreversible binding of multiple molecules via covalent bonds, while "physical cross-linking" and similar expressions refer to the reversible binding of multiple molecules via non-covalent bonds. Furthermore, "physical cross-linking" may or may not be accompanied by chemical binding. Examples of physical cross-linking accompanied by chemical binding include, for example, the binding of multiple molecules via chemical binding (intermolecular forces) such as electrostatic attraction (ion-to-molecule interaction), hydrogen bonding, dipole interaction, and van der Waals forces. Furthermore, examples of physical cross-linking without chemical binding include mechanical adhesion and entanglement of molecules.

[0027] The culture medium disclosed herein can be based on a so-called basal medium, or on a culture medium appropriately formulated according to the cells or other components of the cultured object. As for the basal medium, there are no particular limitations as long as it is a commercially available basal medium; examples include MEM, α-MEM, DMEM, RPMI-1640, and Hams F12.

[0028] Cell culture vector

[0029] The culture medium disclosed herein can be used in the cell culture methods detailed later. As one embodiment, the culture medium of this disclosure can be used in cell culture methods together with cell culture carriers, particularly cell culture microcarriers. The cell culture carriers comprise polysaccharides cross-linked by divalent or higher cations. There are no particular limitations on the polysaccharides used, as long as the effects of this invention are achieved; examples include alginic acid, galacturonic acid, carrageenan, gellan gum, and pectin. These polysaccharides have two or more carboxyl groups, which can form cross-linked structures with divalent or higher cations. Alginic acid is particularly preferred among these polysaccharides. That is, the cell culture carrier preferably comprises polysaccharides cross-linked by divalent or higher cations, including alginic acid. Therefore, in a preferred embodiment, the cell culture carrier is a cell culture microcarrier containing alginic acid as a polysaccharide. Hereinafter, this disclosure will be described using a cell culture microcarrier containing alginic acid cross-linked by divalent or higher cations as an example.

[0030] For alginate crosslinked by divalent or higher cations, for example, droplets containing alginate can be reacted with an aqueous solution containing divalent or higher cations, thereby forming alginate microparticle gels through crosslinking of the alginate by divalent or higher cations. Furthermore, the crosslinking of alginate by divalent or higher cations is a reversible crosslinking (physical crosslinking accompanied by chemical bonding) based on the electrostatic attraction between the anionic portion (e.g., carboxyl group) of the alginate molecule and the divalent or higher cation.

[0031] In cell culture microcarriers, alginate is cross-linked via divalent or higher cations. When using the culture medium disclosed herein, alginate is decross-linked by using a substance that competes with and inhibits the cross-linking of alginate via divalent or higher cations, resulting in the dissolution of the cell culture microcarriers. Examples of such cross-linking inhibitors include chelating agents and monovalent cations. These cross-linking inhibitors can be used alone or in combination of two or more.

[0032] Examples of chelating agents commonly used in cell culture include ethylenediaminetetraacetic acid (EDTA), ethylene glycol ether diaminetetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), N'-(2-hydroxyethyl)ethylenediamine-N,N,N'-triacetic acid (HEDTA), and nitrotriacetic acid (NTA). Among these chelating agents, EDTA is particularly preferred. Furthermore, the concentration of the chelating agent in the case of dissolving microcarriers for cell culture is not particularly limited and can be appropriately set according to the type of chelating agent and the type of solvent used to dissolve it. For example, the concentration of the chelating agent can be 0.5–20 mM, 0.75–15 mM, or 1–10 mM.

[0033] There are no particular limitations on the type of monovalent cation; examples include sodium ions and potassium ions. A monovalent cation can be used by dissolving it in a solvent that does not contain a monovalent cation, or by dissolving it in a solvent that contains a monovalent cation. Furthermore, a monovalent cation can be used in a buffer solution containing a monovalent cation (e.g., PBS). There are no particular limitations on the concentration of the monovalent cation in the case of dissolving microcarriers for cell culture; it can be appropriately set according to the type of monovalent cation, the type of solvent used to dissolve the monovalent cation, etc. For example, the concentration of the monovalent cation can be 10–500 mM, 30–300 mM, 50–200 mM, etc.

[0034] Droplets containing alginate can be formed by extruding alginate in droplets from a fine nozzle into air or oil, and then mixing the droplets with an aqueous solution containing divalent or higher cations to form gelled microparticles (i.e., microcarriers for cell culture). For example, by forming droplets of sodium alginate and mixing the droplets with an aqueous solution of calcium chloride, microcarriers for cell culture containing alginate cross-linked by calcium ions can be formed.

[0035] The alginate constituting the cell culture microcarrier is bound by cross-linking with divalent or higher cations as described above. This can be confirmed by confirming that gel particles are formed when water is added to the cell culture microcarrier in a dry state, and then the gel particles dissolve when EDTA is added.

[0036] In addition to alginate linked by divalent or higher cations as described above, cell culture microcarriers may also contain alginate linked by physical cross-linking. The physical cross-linking used to form such alginate may be accompanied by chemical bonding or not, but physical cross-linking without chemical bonding is preferred (e.g., adhesion or entanglement of alginate particles). Therefore, in one embodiment, the cell culture microcarrier, as alginate, comprises both alginate linked by divalent or higher cations and alginate linked by physical cross-linking.

[0037] Furthermore, in the preparation of the culture medium of this disclosure, when dispersing the cell culture microcarrier in the culture medium, in order to properly maintain the cross-linking structure between the alginate molecules constituting the cell culture microcarrier, it is preferable to include a specific amount of sodium ions and calcium ions in the culture medium.

[0038] The divalent or higher cations constituting the microcarriers for cell culture are not particularly limited, but are preferably selected from one or more of the following: calcium ions, magnesium ions, divalent iron ions, trivalent iron ions, divalent silver ions, trivalent silver ions, copper ions, zinc ions, selenium ions, and other essential trace element ions (manganese ions, chromium ions, cobalt ions, etc.), with calcium ions being more preferred.

[0039] Cell culture microcarriers may contain components other than alginate and divalent or higher cations (i.e., other components). As other components, there are no particular limitations as long as the effects of this disclosure are achieved; examples include cell adhesion substances (scaffold proteins such as gelatin, collagen, laminin, elastin, proteoglycans, fibronectin, and hypotene, peptides and domains having their activities (e.g., RGD peptide, laminin E8, etc.), and serum, etc.). Cell culture microcarriers preferably further contain cell adhesion substances as other components, and gelatin is particularly preferred. As gelatin, gelatin conforming to the specifications of the Japanese Pharmacopoeia, purified gelatin specifications, sterile specifications, microbial limit specifications, purity specifications, or viscosity specifications is preferred; gelatin conforming to the specifications of the Japanese Pharmacopoeia or purified gelatin is particularly preferred. By further containing cell adhesion substances in the cell culture microcarriers, the adhesion of cells to the cell culture microcarriers is improved, resulting in high-efficiency cell culture.

[0040] Gelatin is a substance with high cell adhesion, easy solubility, and low cost. Gelatin can be cross-linked or not. However, if irreversible cross-linking such as chemical cross-linking occurs in gelatin, the microcarriers used for cell culture will be difficult to dissolve with chelating agents after cell culture. Therefore, gelatin used in cell culture microcarriers is preferably not chemically cross-linked or otherwise irreversibly cross-linked.

[0041] On the other hand, cross-linking of gelatin enhances its adsorption to alginate microparticle gels, making it preferable for cell culture. Therefore, from this perspective, cross-linking of gelatin is preferred, and from the viewpoint of the solubility of the microcarriers for cell culture, reversible cross-linking, such as physical cross-linking, is particularly preferred. Physical cross-linking may or may not involve chemical bonding. Furthermore, gelatin may contain both physical and chemical cross-linking as described above. Here, the cross-linking conditions in gelatin are preferably adjusted to achieve a weak cross-linking that allows for easy dissolution of the microcarriers for cell culture after culture. Methods for cross-linking gelatin can be conventionally known methods for molecular cross-linking, specifically including thermal cross-linking, ultraviolet cross-linking, and radiation cross-linking. In addition to gelatin, the cell-adhesive material may include scaffold proteins such as collagen, laminin, elastin, proteoglycans, fibronectin, and hylocinin, peptides or domains with their active sequences (e.g., RGD peptide, laminin E8, etc.), or serum. In this case, it is preferable to contain the microcarrier for cell culture without irreversible cross-linking, so as not to hinder the dissolution of the microcarrier for cell culture by chelating agents or the like after culture.

[0042] In cases where cell culture microcarriers contain gelatin, for example, gelatin can be incorporated into the cell culture microcarriers by impregnating alginic acid particles cross-linked by divalent or higher cations into a gelatin-containing solution. The concentration of gelatin in the solution impregnating the cell culture microcarriers is not particularly limited; however, a higher gelatin concentration results in better swelling in the culture medium and also better cell adhesion and proliferation, which is therefore preferred. The gelatin concentration in the solution impregnating the cell culture microcarriers is preferably more than 0.1% by mass, more preferably more than 0.5% by mass, further preferably more than 1% by mass, even more preferably more than 2% by mass, and particularly preferably more than 4% by mass. When using 1% by mass of alginic acid in the cell culture microcarriers, the gelatin concentration can be increased to a maximum of 99% by mass. Since a higher gelatin concentration results in higher viscosity, from the viewpoint of ease of solution handling, the gelatin concentration in the solution impregnating the cell culture microcarriers is preferably 20% by mass or less, more preferably 15% by mass or less, and even more preferably 10% by mass or less. Furthermore, the solvent described above is not particularly limited, but water is preferred.

[0043] In cases where dried cell culture microcarriers (dried cell culture microcarriers) are used to swell as microcarriers for cell culture, gelatin is preferably contained within the cell culture microcarrier, thereby allowing the dried cell culture microcarriers to swell well in the culture medium. This is because the presence of gelatin between the alginate molecules constituting the dried cell culture microcarriers inhibits the formation of cross-links between the alginate molecules within the dried cell culture microcarriers. Here, when preparing dried cell culture microcarriers, since alginate is negatively charged, if gelatin is positively charged, it may sometimes form insoluble complexes that are difficult to dissolve. Therefore, it is preferable to prepare the dried cell culture microcarriers in a negatively charged state. For this purpose, it is preferable to use gelatin with an acidic isoelectric point, such as gelatin treated with alkali, as gelatin is negatively charged in a neutral state. Furthermore, when using gelatin with an isoelectric point that is alkaline, such as acid-treated gelatin, since gelatin is positively charged in a neutral state, it is preferable to increase the hydroxide ion concentration of the solution used in the preparation of the dried microcarriers for cell culture, etc., to prepare the dried microcarriers for cell culture while making the gelatin negatively charged. Here, since the isoelectric point of acid-treated gelatin is 7 to 9, a pH of 9 or higher is preferred, and a pH of 9 to 13 is particularly preferred. Furthermore, when using cell-adhesive substances other than gelatin, it is also preferable to prepare the dried microcarriers in a negatively charged state. Moreover, from the viewpoint of cell adhesion, it is preferable that at least a portion of the gelatin contained in the dried microcarriers for cell culture is exposed on the surface of the cell culture microcarriers. Furthermore, it is preferable that at least a portion of the gelatin contained in the dried microcarriers for cell culture is bound to the alginate constituting the cell culture microcarriers.

[0044] Furthermore, in dried cell culture microcarriers, compared to undried cell culture microcarriers, their volume decreases and they condense, resulting in a tighter adhesion between the alginic acid components of each dried cell culture microcarrier, as well as between the alginic acid and other components (e.g., cell-adhesive substances such as gelatin). For example, in cases where the dried cell culture microcarriers contain cell-adhesive substances such as gelatin, drying creates physical cross-links between the alginic acid and the cell-adhesive substances, resulting in a strong bond between the two, which is believed to improve cell adhesion.

[0045] Furthermore, dried microcarriers for cell culture, being in a dry state with moisture removed, offer advantages such as high preservation stability and ease of sterilization. Typically, when microcarriers contain a significant amount of liquid (i.e., in a swollen state), the liquid components react chemically with the components constituting the microcarrier (e.g., hydrolysis), resulting in microcarrier decomposition. Conversely, by placing microcarriers in a substantially liquid-free state (i.e., a dry state), chemical reactions between the liquid components and the components constituting the microcarrier can be inhibited, thus suppressing microcarrier decomposition. Moreover, when microcarriers contain a significant amount of liquid, bacteria and other microorganisms may be introduced, potentially leading to microbial proliferation. Since cell culture microcarriers are used in cell culture applications, the potential for microbial proliferation and contamination of target cells by a large number of microorganisms means that conventional antibiotics used in cell culture may not be sufficient to kill or inhibit the proliferation of these microorganisms. On the other hand, dry microcarriers for cell culture, by being in a state that substantially does not contain liquid components, can inhibit the reproduction of microorganisms via liquid components. As a result, they can inhibit the mixing of microorganisms into target cells. These microorganisms can be effectively killed or their proliferation inhibited by the use of general antibiotics during cell culture.

[0046] Therefore, dried microcarriers for cell culture are preferred as microcarriers for cell culture. That is, it is preferable to use dried microcarriers for cell culture by adding them to a swelling solution (e.g., culture medium, water, or other liquid) to swell them and thus create microcarriers for cell culture.

[0047] Furthermore, when impregnating alginate (alginic acid microparticle gel) cross-linked with divalent or higher cations in a solution containing gelatin and then drying it to form dried microcarriers for cell culture, it is preferable to replace the outer layer of the alginate cross-linked with divalent or higher cations with a solvent containing gelatin (preferably water) with a solvent that is not readily available for gelatin before the drying step. This ensures that the gelatin-impregnated alginate cross-linked with divalent or higher cations is surrounded by the solvent that is not readily available for gelatin before drying. This process removes most of the unwanted gelatin from the outer layer of the alginate cross-linked with divalent or higher cations, allowing the gelatin to be sealed inside the microcarriers during drying while preventing the microcarriers from agglomerating together. Specifically, for example, alginic acid cross-linked with divalent or higher cations can be impregnated in an aqueous gelatin solution, allowing the gelatin to soak into the alginic acid cross-linked with divalent or higher cations. Then, the gelatin on the outside of the alginic acid cross-linked with divalent or higher cations is agglomerated (nanoparticle-sized) by ethanol (a poor solvent for gelatin), and washed away to remove the outer gelatin. The alginic acid cross-linked with divalent or higher cations is then dried while surrounded by ethanol. The step of drying the alginic acid microparticle gel impregnated in a solvent containing gelatin is not particularly limited as long as the desired effect of this disclosure is achieved. Vacuum drying, freeze drying, or dry heat drying, or a combination of these methods, is preferred. Vacuum drying preferably takes 30 minutes or more. Dry heat drying preferably takes 100–200°C, more preferably 120–160°C, and for 0.5–6 hours. Higher drying temperatures and longer drying times result in stronger binding of gelatin and gelatin in the microcarrier through physical bonding (physical cross-linking) such as mechanical adhesion and adsorption, and chemical bonding (chemical cross-linking) caused by the Maillard reaction between alginic acid and gelatin. However, when using microcarriers, they are difficult to dissolve using chelating agents, proteolytic enzymes, etc. Therefore, excessively high temperatures or excessively long drying times are not preferred.

[0048] When using dried microcarriers for cell culture, the swelling ratio of the dried microcarriers for cell culture is preferably 214-265%. By ensuring the swelling ratio of the dried microcarriers for cell culture is within this range, the swollen dried microcarriers (i.e., cell culture microcarriers) can maintain a spherical or near-spherical shape without disintegrating. As a result, cells can be cultured with high efficiency. Here, the "swelling ratio" of the dried microcarriers for cell culture can be determined as follows: Prepare dried microcarriers for cell culture and a 24-well plate (product number: MS-90240, manufactured by Sumitomo Bakelite Co., Ltd.) with low cell adsorption treatment. Add 2 mg of dried microcarriers for cell culture and 1 mL of culture medium used in cell culture to one well of the 24-well plate, and allow it to swell for at least 2 hours at a temperature of 37°C and a CO2 concentration of 5%. Then, more than 80 microcarriers for cell culture obtained therefrom were observed using a phase-contrast microscope. The average value of the equivalent diameter of equal-volume spheres was used as the average particle size, which was then used as the average particle size of the dried microcarriers for cell culture after swelling. On the other hand, 2 mg of dried microcarriers for cell culture were added to one well of a 24-well plate, and more than 80 samples were observed using a phase-contrast microscope. The average value of the equivalent diameter of equal-volume spheres was used as the average particle size, which was then used as the average particle size of the dried microcarriers for cell culture before swelling. Then, according to the following formula:

[0049] The swelling rate of the dried microcarriers for cell culture is calculated by dividing the average particle size of the dried microcarriers for cell culture before swelling by (average particle size of the dried microcarriers for cell culture before swelling) by 100.

[0050] Furthermore, since dried microcarriers for cell culture are used as cell culture carriers, the larger their size during use (i.e., after swelling), the larger their surface area, allowing for the adhesion and culture of more cells. Specifically, the size of the swollen dried microcarriers for cell culture is preferably greater than 10 μm, which is approximately the size of the cells. On the other hand, if the size of the swollen dried microcarriers for cell culture is too large, the sedimentation rate is too fast during suspension culture, making cell culture difficult. Specifically, the size of the swollen dried microcarriers for cell culture is preferably 1 mm or less. Therefore, the size of the dried microcarriers for cell culture during use (i.e., after swelling) is not particularly limited, but 1 mm or less is preferred, more preferably 0.6 mm or less, and even more preferably 0.4 mm or less. On the other hand, the size of the dried microcarriers for cell culture during use (i.e., after swelling) is not particularly limited, but 0.19 mm or more is preferred, more preferably 0.1947 mm or more, even more preferably 0.2 mm or more, and particularly preferably 0.21 mm or more. The dried microcarriers for cell culture are in a dry state, but in cases where they are used in cell culture, they are usually used in a swollen state. Therefore, their size increases when used (i.e., after swelling) compared to their dry state. Thus, in order to ensure that the size is within the aforementioned range when used, the size of the dried microcarriers for cell culture (i.e., before swelling) is preferably greater than 4.5 μm, further preferably 520 μm or less, more preferably 310 μm or less, and even more preferably 210 μm or less.

[0051] Sodium ions and calcium ions

[0052] The culture medium disclosed herein contains sodium and calcium ions. In one embodiment, the mass concentration of sodium ions in the culture medium of this disclosure is 2600–3600 ppm, preferably 2900–3600 ppm. By maintaining the sodium ion mass concentration in the culture medium within the above range, the osmotic pressure balance of the cells can be well maintained during cell culture, and osmotic pressure-induced cell damage can be inhibited. The mass concentrations of sodium and calcium ions in the culture medium of this disclosure, and the ratio of calcium ion mass to sodium ion mass, can be adjusted by adding sodium and / or calcium monomers, compounds containing sodium and / or calcium, to the culture medium, or by adding a basal culture medium containing sodium and / or calcium to the culture medium. Furthermore, in this disclosure, "ppm" means "mg / L".

[0053] In the culture medium disclosed herein, the lower limit of the ratio of calcium ion mass to sodium ion mass is 2.8% by mass, preferably 2.9% by mass, more preferably 3% by mass, and particularly preferably 3.1% by mass. On the other hand, the upper limit of the ratio of calcium ion mass to sodium ion mass is 48.5% by mass, preferably 47% by mass, more preferably 46% by mass, further preferably 45% by mass, even more preferably 44% by mass, and particularly preferably 43.8% by mass. Furthermore, the range of the ratio of calcium ion mass to sodium ion mass is 2.8% by mass or more and 48.5% by mass or less, preferably 2.8% by mass or more and 47% by mass or less, more preferably 2.8% by mass or more and 46% by mass or less, further preferably 2.9% by mass or more and 47% by mass or less, even more preferably 3% by mass or more and 46% by mass or less, and particularly preferably 3.1% by mass or more and 43.8% by mass or less. By maintaining the ratio of sodium ion mass to calcium ion mass in the culture medium within the above-mentioned range, the shape of the cell culture microcarrier is stably maintained, resulting in high-efficiency cell culture.

[0054] In one embodiment, the ratio of calcium ion mass to sodium ion mass in the culture medium of this disclosure is adjusted to 2.8% by mass or more and 48.5% by mass or less, preferably 3% by mass or more and 30% by mass or less, more preferably 3% by mass or more and 29% by mass or less, even more preferably 3.1% by mass or more and 15.3% by mass or less, even more preferably 3.1% by mass or more and 10% by mass or less, and particularly preferably 3.1% by mass or more and 8.8% by mass or less. By maintaining the ratio of sodium ion mass to calcium ion mass in the culture medium within the above-mentioned range, the shape of the cell culture microcarrier is stably maintained, enabling cell culture with particularly high efficiency.

[0055] The culture medium disclosed herein may contain components other than sodium and calcium ions as described above, provided that it does not impair the effectiveness of the present disclosure. Other components are not particularly limited to those commonly used in cell culture, such as buffers, sugars, amino acids, vitamins, carbohydrates, fatty acids, antibiotics, inorganic salts, phenol red, proteins, peptides, serum, nucleic acid bases, and glucose. Specifically, buffers (e.g., HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid)), amino acids, and glucose are used to adjust the osmotic pressure of the cells during the cell culture process.

[0056] <Cultivation Methods>

[0057] According to another aspect of this disclosure, a method (also referred to herein as "the method of this disclosure," etc.) is provided for culturing cells.

[0058] It includes a culture step of culturing the cells in a culture medium.

[0059] The ratio of calcium ion mass to sodium ion mass in the culture medium is greater than 2.8% by mass and less than 48.5% by mass.

[0060] Cultivation steps

[0061] The method of this disclosure includes the step of culturing cells in a culture medium (i.e., the culture step). As the "culture medium" used in the culture step, for example, the "culture medium of this disclosure" described above can be used.

[0062] In one embodiment, the method of this disclosure uses cell culture microcarriers containing alginate, preferably cell culture microcarriers containing alginate cross-linked with divalent or higher cations, for the cell culture step. The method of this disclosure can stably maintain the shape of cell culture microcarriers, particularly those containing alginate, by using a culture medium with a calcium ion mass ratio to sodium ion mass within a specific range, resulting in high-efficiency cell culture. The reason for stably maintaining the shape of cell culture microcarriers containing alginate by using a culture medium with a calcium ion mass ratio to sodium ion within a specific range is as follows: Alginate has water-soluble properties by ionic cross-linking with sodium ions to form sodium alginate. On the other hand, if calcium ions are applied to an aqueous solution of sodium alginate, the alginate constituting sodium alginate rapidly undergoes ionic cross-linking with calcium ions (i.e., sodium ions cross-linked with alginate ions are replaced by calcium ions), forming water-insoluble calcium alginate (gelation). Here, if the calcium ions constituting calcium alginate undergo an ion exchange reaction with sodium ions, the calcium alginate gel becomes partially or completely soluble in water. As a result, the shape of the cell culture microcarrier becomes unstable and may break down. Therefore, in order to stably maintain the gelled calcium alginate, it is necessary to suppress the ion exchange reaction between the calcium ions constituting the calcium alginate gel and the sodium ions present in the system containing the calcium alginate gel. Therefore, the ratio of sodium ions to calcium ions present in the system containing the calcium alginate gel becomes important. In the method of this disclosure, the ratio of calcium ion mass to sodium ion mass in the culture medium is adjusted to 2.8% by mass or more and 48.5% by mass or less. In particular, by setting this ratio to 2.8% by mass or more, the aforementioned ion exchange reaction between calcium ions and sodium ions is suppressed, and the shape of the cell culture microcarrier is considered to be stably maintained. In the case of cell culture using cell culture microcarriers containing alginate, the culture medium is generally mixed and cultured while being stirred and shaken, thus applying a certain mechanical load to the cell culture microcarrier. Therefore, maintaining the shape of the microcarriers used for cell culture stably and preventing their collapse under such conditions is extremely important from the perspective of cell culture efficiency. On the other hand, by keeping the ratio of calcium ions to sodium ions in the culture medium below 48.5% by mass, toxicity to cells during cell culture can be suppressed, and cell proliferation efficiency can be improved.

[0063] The culture conditions in the culture step can be appropriately set according to the cell type, the composition of the culture medium, and the type / volume of the culture container. Furthermore, the culture step can be carried out in a static state, or it can be performed by stirring the culture medium to suspend the cell culture microcarriers. Suspension culture is preferred to culture more cells using the cell culture microcarriers.

[0064] Recycling steps

[0065] The method of this disclosure may further include a recovery step after the above-described culture step to recover the cultured cells. In the case where the culture step is performed using cell culture microcarriers containing alginate cross-linked with divalent or higher cations, the recovery step can be performed by adjusting the ratio of calcium ion mass to sodium ion mass in the culture medium to less than 2.8% by mass. By adjusting the ratio of calcium ion mass to sodium ion mass in the culture medium to the above-described range, the cell culture microcarriers can be soluble, thus allowing separation of cells from the cell culture microcarriers and easy recovery of cells. To adjust the ratio of calcium ion mass to sodium ion mass, the above-described sodium ion and / or calcium ion concentrations can be used in the culture medium of this disclosure. Furthermore, the method for adjusting the ratio of calcium ion mass to sodium ion mass in the culture medium is not particularly limited as long as the effects of this disclosure are achieved; for example, it can be adjusted by the following method. First, the cell culture microcarriers with adhered cells in culture are allowed to sink to the bottom of the culture vessel, and the supernatant is removed while retaining the adhered cells. Then, a washing solution is added to the residue after removing the supernatant. Examples of washing solutions include PBS. Then, the supernatant is removed using cell culture microcarriers that retain the washed cell adhesion. A recovery solution is then added to the remaining portion after supernatant removal. Examples of recovery solutions include TrypLE Select (trademark), EDTA / PBS solution, etc. Alternatively, the recovery step can be performed by adding a chelating agent to the culture medium. By adding a chelating agent to the culture medium, the cell culture microcarriers react with the chelating agent, removing divalent or higher cations of cross-linked alginate from the alginate, thus making the cell culture microcarriers soluble.

[0066] EDTA is preferably used as the chelating agent in the recovery step. Furthermore, adding alginate-degrading enzymes or proteolytic enzymes in addition to the chelating agent can hydrolyze alginate and gelatin, allowing for faster dissolution of the cell culture microcarriers. On the other hand, from the viewpoint of minimizing cell damage, it is preferable not to use proteolytic enzymes. Moreover, to minimize the amount of added substances from both a safety and cost perspective, it is preferable not to use alginate-degrading enzymes. Based on the above, dissolving the cell culture microcarriers using only a chelating agent is most preferred. Examples of proteolytic enzymes include proteases, collagenases, and trypsin.

[0067] swelling steps

[0068] The method disclosed herein can be used as a cell culture microcarrier using the aforementioned dried cell culture microcarriers with respect to the culture medium of this disclosure. Therefore, in the case where dried cell culture microcarriers are used as cell culture microcarriers, the method of this disclosure preferably further includes a swelling step to prepare the cell culture microcarriers by contacting them with a swelling solution to swell them before the aforementioned culture step. Since dried cell culture microcarriers have advantages such as high preservation stability and the ability to inhibit contamination through sterilization, by using such dried cell culture microcarriers to swell in the swelling step, cells can be cultured with high efficiency.

[0069] The method for swelling dried microcarriers for cell culture is not particularly limited as long as it can maintain the stability of the microcarriers before cell culture. Examples include adding the dried microcarriers to a swelling solution with a calcium ion mass ratio of 2.8% to 48.5% (by mass) or more but less than the sodium ion mass ratio. Specifically, when using dried microcarriers for cell culture containing calcium alginate, by using a swelling solution with a calcium ion mass ratio of 2.8% to 48.5% (by mass) or more but less than the sodium ion mass ratio, the sodium ions in the swelling solution act to a degree that prevent the difficult-to-swell calcium alginate portion of the dried microcarriers from undergoing ion exchange reactions with the calcium cross-linked with alginate ions. As a result, the calcium alginate can be appropriately unwound, causing the dried microcarriers to swell. Examples of swelling solutions with a calcium ion mass ratio of 2.8% to 48.5% (by mass) or less but less than the sodium ion mass ratio include those containing calcium chloride added to physiological saline (9 g / L sodium chloride aqueous solution) and those containing calcium chloride added to the culture medium. In cases where a swelling solution with a calcium ion mass ratio of 2.8% to 48.5% by mass or less relative to sodium ions is used for swelling of dried microcarriers for cell culture, using a swelling solution containing added calcium chloride to physiological saline is preferable from a cost perspective. From the viewpoint of simplifying the method of this disclosure, it is preferable to use a swelling solution with the same composition as the culture medium used to culture the actual cells to swell the dried microcarriers for cell culture. Therefore, it is unnecessary to prepare a separate swelling solution for the swelling step.

[0070] As the target cells of the method of this disclosure, adhesive cells that can be cultured on the surface of cell culture microcarriers are preferred. Examples of such cells include, for instance, hepatocytes (parenchymal cells of the liver), Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, epidermal cells such as epidermal keratinocytes, tracheal epithelial cells, digestive tract epithelial cells, cervical epithelial cells, corneal epithelial cells, mammary cells, pericytes, myoblasts, myotube cells, satellite cells, smooth muscle cells, cardiomyocytes, kidney cells, pancreatic islet cells, peripheral nerve cells, optic nerve cells, chondrocytes, and osteocytes. These cells can be primary cells directly collected from tissues or organs, or cells that have been passaged several times. Furthermore, these cells can be any of the following: undifferentiated embryonic stem cells, iPS cells, somatic stem cells such as mesenchymal stem cells with differentiation capacity, unipotent stem cells such as vascular endothelial progenitor cells with single differentiation capacity, or cells that have completed differentiation. The target cells of the method disclosed herein can further be CHO cells, 293 cells, 3T3 cells, Vero cells, MRC5 cells, HeLa cells, HEK293 cells, hybridomas, etc., and cell lines established from them, which are widely used as cell matrices for preparing biological drugs and viral vectors. In addition, the target cells of the method disclosed herein can be cultured as a single type of cell, or two or more types of cells can be co-cultured. By culturing such cells according to the method disclosed herein, medical products and foods with expected therapeutic effects can be prepared. Specifically, the cells obtained by the method disclosed herein can be used as pharmaceutical compositions (i.e., pharmaceutical compositions containing cells). Such pharmaceutical compositions can be prepared, for example, using cells obtained by the method disclosed herein, and pharmaceutically acceptable additives (e.g., excipients, solvents, cryopreservation solutions, etc.) as needed, using common techniques for preparing pharmaceutical compositions. As a cryopreservation solution, for example, a liquid containing a cell protectant can be used. Examples of cell protectants include dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, sericin, glycerol, etc. A pharmaceutical composition can be prepared by suspending and mixing cells obtained by the method of this disclosure in the cryopreservation solution.

[0071] Example

[0072] The present disclosure will be described in more detail below through embodiments, but the present disclosure is not limited to the following embodiments.

[0073] Experimental Example 1: Study on the concentrations of sodium and calcium ions in the culture medium

[0074] Prepare the culture medium and cell culture microcarriers according to the following procedures.

[0075] <Preparation of Culture Medium>

[0076] Prepare α-MEM (product number: 21444-05, manufactured by Nacalai Tesque Co., Ltd.), and adjust it to 10% FBS (product number: 26140-079, manufactured by GIBCO) and 20 ng / mL β-FGF (basic fibroblast growth factor) (hereinafter referred to as "basal medium"). Further, prepare 500 mM calcium chloride aqueous solution, adding it to 1 / 800, 1 / 400, 1 / 200, 1 / 100, 1 / 50, 1 / 25, 1 / 20, 1 / 15, 1 / 12.5, 1 / 6.25, and 1 / 3.125 of the volume relative to the basal medium, preparing 11 different culture media with varying calcium chloride concentrations. Additionally, prepare basal medium without added calcium chloride aqueous solution. For these 12 culture media, the concentrations of calcium and sodium ions were measured within 1 hour of preparation at 25 ± 1 °C. Furthermore, for α-MEM, unopened products or those within their shelf life and retaining more than 70% of the original volume of the culture medium when it was purchased are used, so that changes in the pH of the culture medium can be disregarded. In addition, each measurement is performed within one hour of the preparation of each culture medium.

[0077] Calcium ion concentration was measured using a compact calcium ion meter LAQUAtwinCa-11 (product number: 3200689161, manufactured by Horiba Manufacturing Co., Ltd.). Here, for each of the 10 types of culture media containing 1 / 800, 1 / 400, 1 / 200, 1 / 100, 1 / 50, 1 / 25, 1 / 20, 1 / 15, and 1 / 12.5 of calcium chloride aqueous solution, and for each culture media without calcium chloride aqueous solution, the calcium ion concentration was measured after first-point calibration at 150 ppm using calcium ion standard solution (150 ppm) Y051L (product number: 3200457728, manufactured by Horiba Manufacturing Co., Ltd.), and second-point calibration at 2000 ppm using calcium ion standard solution (2000 ppm) Y051H (product number: 3200457727, manufactured by Horiba Manufacturing Co., Ltd.). Furthermore, for the two culture media containing 1 / 6.25 and 1 / 3.125 of the calcium chloride aqueous solution, respectively, the calcium ion concentration was measured after first-point calibration using calcium ion standard solution (2000 ppm) Y051H (product number: 3200457727, manufactured by Horiba Manufacturing Co., Ltd.) at 2000 ppm, and second-point calibration using a separately prepared 200 mM calcium chloride aqueous solution at 8000 ppm. The measurement results are shown in Table 1.

[0078] Sodium ion concentration was measured using a compact sodium ion meter, LAQUAtwinNa-11 (Product No.: 3200689159, manufactured by Horiba Corporation). For each of the 12 culture media mentioned above, sodium ion concentration was measured after first-point calibration at 2000 ppm using a sodium ion standard solution (2000 ppm) Y022H (Product No.: 3200457723, manufactured by Horiba Corporation), and second-point calibration at 3500 ppm using a separately prepared 9 g / L sodium chloride aqueous solution. The measurement results are shown in Table 1.

[0079] [Table 1]

[0080]

[0081] As shown in Table 1, 12 different culture media with varying ratios of calcium ion mass to sodium ion mass were prepared.

[0082] <Preparation of dried microcarriers for cell culture>

[0083] Prepare 1% aqueous solutions of sodium alginate (product number: 194-13321, manufactured by Fujifilm Corporation) and 1% aqueous solutions of calcium chloride (product number: 038-24985, manufactured by Fujifilm Corporation).

[0084] Sodium alginate aqueous solution was added dropwise to calcium chloride aqueous solution using a 32G injection needle to prepare a calcium alginate microparticle gel with a diameter of approximately 200 μm (measured by optical microscopy) (alginic acid crosslinked via calcium ions) (Reference) Figure 1 The microparticle gel was washed with 70% ethanol and water, and recovered using a cell sieve (product number: 352340, manufactured by FALCON). The microparticle gel was then immersed in a 4% (w / w) aqueous solution of alkali-treated gelatin (product number: G9391-100G, manufactured by Sigma-Aldrich) that had been autoclaved, and placed at 20°C for at least 2 hours to allow the gelatin to penetrate the interior of the microparticle gel.

[0085] Here, the microparticle gel size remained at approximately 200 μm in diameter, and no shape change was observed. After recovering the microparticle gel using a cell sieve, it was further purified by immersing it in ethanol and then using the cell sieve again to remove the gelatin from the outside of the microparticle gel and encapsulate the gelatin within it. Under an optical microscope, the calcium alginate microparticle gel before gelatin encapsulation was colorless, while the gelatin aqueous solution was slightly yellow. However, after being surrounded by ethanol and encapsulated with gelatin, the interior of the microparticle gel was slightly yellow, while the exterior remained colorless, thus confirming that the gelatin was encapsulated within the microparticle gel.

[0086] After vacuum drying in this state, water is removed by dry heat drying at 150°C for 2 hours, and the size is reduced to approximately 90 μm (measured by optical microscopy), yielding powdered dried microcarriers for cell culture (reference). Figure 2 Here, it is a white powder immediately after vacuum drying, and turns brown after dry heat drying.

[0087] <Preparation of Cell Culture Plates>

[0088] Using the culture media and dried microcarriers for cell culture prepared according to the above procedure, prepare cell culture plates according to the following procedure. Specifically, prepare dried microcarriers for cell culture and 24-well plates with low cell adsorption treatment (product number: MS-90240, manufactured by Sumitomo Bakelite Co., Ltd.). Add 2 mg of dried microcarriers for cell culture and 1 mL of culture media from test zones 1 to 12 to one well of the 24-well plate. Then, incubate at 37°C and 5% CO2 concentration to allow the dried microcarriers for cell culture to swell for at least 2 hours to prepare the cell culture plate.

[0089] <Cell Culture>

[0090] In cell culture plates prepared according to the above procedure, human adipose-derived stem cells (product number: PT-5006, Lonza Corporation) were seeded at a density of 5.6 × 10⁶ cells per well. 4 Each well contained 2 mL of culture medium and was cultured for 7 days at 37°C and 5% CO2. Then, on days 1, 2, 3, 6, and 7, the suspension containing cells and cell culture microcarriers was gently pipetted from the wells using a 1 mL pipette. On day 7, the culture medium was observed using a phase-contrast microscope to confirm the condition of the cell culture microcarriers. The observation results are shown in Table 2 and... Figure 3 .

[0091] [Table 2]

[0092]

[0093] According to Table 2 and Figure 3 The results show that in the culture media of experimental zones 2 to 12, where the ratio of calcium ion mass to sodium ion mass in the culture medium was 3.1% or more, the cell culture microcarriers maintained a stable spherical or near-spherical shape without breaking down.

[0094] Experimental Example 2: Confirmation of Growth Ratio

[0095] For cell culture microcarriers covering the entire experimental area 2–12 maintaining a spherical or near-spherical shape, live cells were stained with calcein-AM solution (product number: 19177-14, manufactured by Nacalai Tesque Co., Ltd.) and photographed using a fluorescence microscope. Results are shown in… Figure 4 In addition, cell culture was performed using the same culture medium as in Experiment 1, using wells 2-12. Viable cells were not stained, and the supernatant from each well was removed. The microcarriers containing adherent cells were washed with 1 mL of PBS, and the PBS supernatant was removed again. 0.5 mL of TrypLE (Product No.: 12563-029, ThermoFisher Scientific Co., Ltd.) was added, and the microcarriers were dissolved at 37°C for 5 minutes. The cell suspension was then recovered. Basic culture medium was added to the recovered cell suspension to a total volume of 1 mL to prepare the cell suspension. The cell suspension was then analyzed using NucleoCounter NC-202 (Product No.: 970-2020, ChemoMetec Co., Ltd.) under the Count & Viability condition, and the viable cell count was calculated relative to the initial seeding number of 5.6 × 10⁶ cells / well. 4 The ploidy rate of each individual was determined. The results are shown in Table 3.

[0096] [Table 3]

[0097]

[0098] According to Table 3 and Figure 4 The results show that in the culture media of test zones 2 to 9, where the ratio of calcium ion mass to sodium ion mass was 43.8% or less, cell adhesion to the cell culture microcarriers was observed, and cell proliferation reached a level that covered the entire surface of the microcarriers, achieving a proliferation rate of more than 1 times relative to the initial seeding number. In particular, a proliferation rate of more than 3 times was achieved when the ratio of calcium ion mass to sodium ion mass was 29.0% or less, a proliferation rate of more than 5 times was achieved when the ratio was 15.3% or less, and an extremely high proliferation rate of more than 7 times was achieved when the ratio was 8.8% or less.

[0099] Experimental Example 3: Study on the swelling rate of dried microcarriers for cell culture

[0100] Using the culture media and dried microcarriers for cell culture prepared according to the above procedure, the swelling rate of the dried microcarriers for cell culture was studied according to the following procedure. First, dried microcarriers for cell culture and 24-well plates with low cell adsorption treatment (product number: MS-90240, manufactured by Sumitomo Bakelite Co., Ltd.) were prepared. 2 mg of dried microcarriers for cell culture and 1 mL of culture media from test zones 1 to 12 were added to one well of the 24-well plate. The dried microcarriers for cell culture were allowed to swell for more than 2 hours at a temperature of 37°C and a CO2 concentration of 5%.

[0101] Then, the culture medium in each experimental zone was observed using a phase-contrast microscope. The equivalent diameter of at least 80 swollen dried microcarriers for cell culture was measured, and their average value was taken as the average particle size of the swollen dried microcarriers for cell culture. Furthermore, dried microcarriers for cell culture were placed in 24-well plates (product number: MS-90240, manufactured by Sumitomo Bakelite Co., Ltd.), and the equivalent diameter of at least 80 swollen dried microcarriers for cell culture was measured using a phase-contrast microscope. Their average value was taken as the average particle size of the dried microcarriers for cell culture. The average particle size of the dried microcarriers for cell culture was 91 μm. Then, according to the following formula:

[0102] The swelling rate of the dried microcarriers for cell culture in each experimental zone was calculated by dividing the average particle size of the dried microcarriers for cell culture before swelling by (average particle size of the dried microcarriers for cell culture before swelling) by 100. The results are shown in Table 4.

[0103] [Table 4]

[0104]

[0105] According to the results shown in Tables 2 and 4, in the culture media of test zones 2–12 where the swelling ratio of the dried microcarriers for cell culture was 208–259%, the microcarriers maintained a spherical or near-spherical shape throughout the test, remaining stable without disintegration. Furthermore, according to the results shown in Tables 3 and 4, in the culture media of test zones 2–9 where the swelling ratio of the dried microcarriers for cell culture was 214–259%, the microcarriers maintained a spherical or near-spherical shape throughout the test, not only remaining stable without disintegration, but also showing cell adhesion to the microcarriers and cell proliferation to the point of completely covering the surface of the microcarriers, achieving a proliferation rate exceeding 1 times relative to the initial inoculation number. In particular, it is known that when the swelling ratio of the dried microcarriers for cell culture is 223-259%, the microcarriers for cell culture maintain a spherical or near-spherical shape throughout, not only without collapsing and remaining stable, but also achieving a high proliferation rate of more than 5 times relative to the initial inoculation number, and an extremely high proliferation rate of more than 7 times when the swelling ratio is 233-259%.

[0106] Furthermore, generally, the larger the average particle size of the swollen dried microcarriers for cell culture, the larger their surface area, allowing for the adhesion and culture of more cells. For example, in experimental zone 7 of Table 4, the average particle size of the swollen dried microcarriers for cell culture is 196 μm (0.0196 cm). Therefore, if its radius r is 0.0098 cm, the surface area (area where cells can adhere) of each microcarrier is approximately 0.001207 cm². 2 On the other hand, in test area 2 of Table 4, the average particle size of the swollen, dried microcarriers for cell culture is 235 μm (0.0235 cm). Therefore, if its radius r is 0.01175 cm, the surface area of ​​each microcarrier is approximately 0.001735 cm². 2 Compared to experimental zone 7, the surface area of ​​experimental zone 2 is approximately 1.4 times larger; therefore, theoretically, approximately 1.4 times more cells can be cultured. Then, according to the results shown in Table 3, the actual proliferation rate in experimental zone 7 was 3.1 times. Therefore, if the increase in the surface area of ​​the microcarriers were the sole factor, the theoretical proliferation rate in experimental zone 2 should be 4.3 times. However, according to the results shown in Tables 3 and 4, the actual proliferation rate in experimental zone 2 was greater than 4.3 times, thus confirming that the proliferation rate can be increased beyond the effect of increased surface area. These results also suggest that the ratio of calcium ion mass to sodium ion mass is an important factor for increasing cell proliferation rate.

[0107] Experimental Example 4: Study on Potassium Ion Concentration

[0108] Using the culture media and dried microcarriers for cell culture prepared according to the above procedure, the potassium ion concentration in the culture media was studied according to the following procedure. First, for each culture medium in test areas 1-12, potassium ions were measured using a compact potassium ion meter LAQUAtwinK-11 (product number: 3200689160, manufactured by Horiba Corporation). For each culture medium, the potassium ion concentration was measured after first-point calibration at 150 ppm using potassium ion standard solution (150 ppm) Y031L (product number: 3200457720, manufactured by Horiba Corporation) and second-point calibration at 2000 ppm using potassium ion standard solution (2000 ppm) Y031H (product number: 3200457719, manufactured by Horiba Corporation). The measurement results are shown in Table 5.

[0109] [Table 5]

[0110]

[0111] As shown in Tables 1 and 5, the ratio of calcium ion mass to potassium ion mass differed in the culture media of experimental zones 1–12, as did the ratio of calcium ion mass to the sum of sodium and potassium ion masses. Furthermore, based on the results in Tables 1 and 5, in the culture media of experimental zones 2–12, where the ratio of calcium ion mass to potassium ion mass was 37.0% or more, or the ratio of calcium ion mass to the sum of sodium and potassium ion masses was 2.9% or more, and the ratio of calcium ion mass to potassium ion mass was 2208.3% or less, or the ratio of calcium ion mass to the sum of sodium and potassium ion masses was 168.8% or less, the cell culture microcarriers maintained a spherical or near-spherical shape throughout, remaining stable without disintegration. Furthermore, according to the results shown in Tables 3 and 5, in the culture media of test zones 2 to 9 where the mass ratio of calcium ions to potassium ions was 37.0% or more, or the mass ratio of calcium ions to the sum of sodium and potassium ions was 2.9% or more, and the mass ratio of calcium ions to potassium ions was 518.5% or less, or the mass ratio of calcium ions to the sum of sodium and potassium ions was 40.3% or less, the cell culture microcarriers maintained a spherical or near-spherical shape throughout, not only remaining stable without breaking down, but also showing cell adhesion to the cell culture microcarriers, and cell proliferation to the extent that it covered the entire surface of the microcarriers, achieving a proliferation rate of more than 1 times relative to the initial inoculation number. Specifically, it is known that when the mass ratio of calcium ions to potassium ions is 37.0% or more, or the mass ratio of calcium ions to the sum of the masses of sodium and potassium ions is 2.9% or more, and the mass ratio of calcium ions to potassium ions is 346.2% or less, or the mass ratio of calcium ions to the sum of the masses of sodium and potassium ions is 26.8% or less, the cell culture microcarriers maintain a spherical or near-spherical shape throughout, are stable without breaking down, and achieve a proliferation rate of more than 3 times relative to the initial inoculation number. It is further understood that when the ratio of calcium ion mass to potassium ion mass is 37.0% or more, or the ratio of calcium ion mass to the sum of sodium and potassium ion mass is 2.9% or more, and the ratio of calcium ion mass to potassium ion mass is 181.5% or less, or the ratio of calcium ion mass to the sum of sodium and potassium ion mass is 14.1% or less, the cell culture microcarriers maintain a spherical or near-spherical shape throughout, not only remaining stable without breaking down, but also achieving a proliferation rate of more than 5 times relative to the initial inoculation number.It is further understood that when the ratio of calcium ion mass to potassium ion mass is 37.0% or more, or the ratio of calcium ion mass to the sum of sodium and potassium ion mass is 2.9% or more, and the ratio of calcium ion mass to potassium ion mass is 103.7% or less, or the ratio of calcium ion mass to the sum of sodium and potassium ion mass is 8.1% or less, the cell culture microcarriers maintain a spherical or near-spherical shape throughout, not only remaining stable without breaking down, but also achieving an extremely high proliferation rate of more than 7 times relative to the initial inoculation number.

[0112] Experimental Example 5: Study on the changes in sodium ion concentration and calcium ion concentration

[0113] Using the culture media and dried microcarriers for cell culture prepared according to the above procedure, the changes in sodium and calcium ion concentrations in the culture media over time before and after culture were studied according to the following procedure. First, dried microcarriers for cell culture and 24-well plates with low cell adsorption treatment (product number: MS-90240, manufactured by Sumitomo Bakelite Co., Ltd.) were prepared. 2 mg of dried microcarriers for cell culture and 1 mL of culture media from test zones 2 to 9 were added to one well of the 24-well plate. Then, the 24-well plate was allowed to stand at 37°C and 5% CO2 concentration for at least 2 hours to allow the dried microcarriers for cell culture to swell, thus preparing the cell culture plate.

[0114] Human adipose-derived stem cells (product number: PT-5006, manufactured by Lonza Co., Ltd.) were seeded into each cell culture plate prepared according to the above procedure, resulting in a cell count of 5.6 × 10⁶ cells per well. 4 The total volume of culture medium in each well is 2 mL. The culture is then incubated for 1 day at a temperature of 37℃ and a CO2 concentration of 5%.

[0115] After one day of culture, the supernatant of the culture medium was sampled from each well, and the sodium and calcium ion concentrations of each sampled supernatant were determined using the method described above. Furthermore, to avoid the influence of culture medium evaporation on the ion concentrations, the evaporation amount of the culture medium in each well was confirmed by mass measurement before supernatant sampling (i.e., the difference between the mass of the culture medium at the start of culture (immediately after swelling of the dried microcarriers for cell culture) and the mass of the culture medium after one day of culture). Sterile water was added accordingly to the evaporation amount, and the concentrations were then measured. The results of the ion concentration measurements before and after culture are shown in Table 6. Additionally, the ion concentrations at the start of culture (immediately after swelling of the dried microcarriers for cell culture) were the same as those shown in Table 1 above.

[0116] [Table 6]

[0117]

[0118] According to the results shown in Table 6, for any culture medium in experimental zones 2–9, the Ca concentration in the culture medium after one day of incubation was... 2＋ / Na ＋ [mass %] Ca in the culture medium relative to the start of culture (immediately after swelling of the dried microcarriers for cell culture). 2＋ / Na ＋ [Mass%] All values ​​were more than 1 times. These results suggest that by releasing calcium ions contained in the microcarriers into the culture medium, maintaining an appropriate amount of calcium ions inside and outside the microcarriers, and preventing excessive shrinkage / expansion and shape changes in the microcarriers to maintain stability, cells can be cultured with high efficiency.

[0119] Experimental Example 6: Study on the variation of microcarrier particle size

[0120] Using the culture media and dried microcarriers for cell culture prepared according to the above procedure, the changes in the particle size of the microcarriers over time before and after culture were studied according to the following procedure. Specifically, after culturing human adipose-derived stem cells for 1 day, as in Experimental Example 5 above, before sampling the supernatant of each culture medium, the equivalent spherical diameter of more than 80 swollen microcarriers in each culture medium was measured, and their average value was taken as the average particle size of the swollen microcarriers after 1 day of culture (average particle size on day 1). The results of the measurement of the average particle size of the microcarriers before and after culture are shown in Table 7. In addition, the average particle size of the microcarriers at the start of culture (immediately after the swelling of the dried microcarriers for cell culture) is the same as that shown in Table 4 above.

[0121] [Table 7]

[0122]

[0123] As shown in Table 7, for any of the culture media in experimental zones 2–9, the average particle size of the microcarriers after one day of culture was more than double that at the start of culture (immediately after swelling of the dried microcarriers used for cell culture). These results suggest that by releasing calcium ions contained within the microcarriers into the culture medium, maintaining an appropriate amount of calcium ions inside and outside the microcarriers, and moderately increasing the size (average particle size) of the microcarriers while maintaining stability, cells can be cultured efficiently. Furthermore, this suggests that, due to the increased surface area for cell proliferation resulting from the increased size of the microcarriers, a moderate increase in microcarrier size is appropriate from the perspective of efficient cell culture.

[0124] Another aspect of the present invention relates to the following [1] to [9].

[0125] [1] Methods for culturing cells,

[0126] It includes a culture step of culturing said cells in a culture medium using a cell culture carrier containing polysaccharides cross-linked by divalent or higher cations.

[0127] The ratio of calcium ion mass to sodium ion mass in the culture medium is greater than 2.8% by mass and less than 48.5% by mass.

[0128] [2][1] The method described herein, wherein the cell culture carrier is a cell culture microcarrier.

[0129] The method described in [3][1] or [2], wherein the polysaccharide includes alginate.

[0130] The method described in any one of [4][1] to [3], wherein the culture step is performed using a cell culture microcarrier containing alginate cross-linked with divalent or higher cations.

[0131] The method described in any one of [5][1] to [4], wherein the divalent or higher cation includes one or more selected from calcium ions, magnesium ions, ferrous ions, ferric ions, ferric ions, ferrous ions, ferrous silver ions, ferric silver ions, copper ions, zinc ions, selenium ions and other essential trace element ions.

[0132] The method described in [6][4] or [5], wherein the alginic acid crosslinked by divalent or higher cations comprises calcium alginate.

[0133] The method described in any of [7][1] to [6], wherein the mass concentration of sodium ions in the culture medium is 2600 to 3600 ppm.

[0134] The method described in any of [8][1] to [7], wherein the cell culture carrier further comprises a cell adhesion substance.

[0135] [9][8] The method described herein, wherein the cell adhesion substance comprises one or more selected from gelatin, collagen, laminin, elastin, proteoglycan, fibronectin, fibronectin, peptides and domains having their activity, and serum.

[0136] The method described in any one of

[10] [1] to [9] further includes the step of preparing the cell culture carrier by contacting and swelling the dried cell culture carrier with a swelling solution prior to the culture step.

[0137] The method described in any of

[11] [1] to

[10] further includes a recovery step of recovering the cultured cells after the culture step.

[0138] The recovery step includes adjusting the ratio of calcium ion mass to sodium ion mass in the culture medium to less than 2.8 by mass.

[0139] The method described in any one of

[12] [1] to

[11] , wherein the ratio of the mass of calcium ions in the culture medium to the sum of the mass of sodium ions and the mass of potassium ions is 2.9% by mass or more and 168.8% by mass or less.

[0140] The method described in any one of

[13] [1] to

[12] , wherein at the beginning of the culture step, the ratio of the mass of calcium ions to the mass of sodium ions in the culture medium is adjusted to 2.8% by mass or more and 48.5% by mass or less.

[0141] The method described in any one of

[14] [1] to

[13] , wherein the ratio of the mass of calcium ions to the mass of sodium ions in the culture medium after one day from the start of the culture step is more than 1 relative to the ratio of the mass of calcium ions to the mass of sodium ions in the culture medium at the beginning of the culture step.

[0142] The method described in any one of

[15] [1] to

[14] , wherein the average particle size of the cell culture carrier after 1 day from the start of the culture step is more than 1 times the average particle size of the cell culture carrier at the start of the culture step.

[0143] The method described in any one of

[16] [1] to

[15] , wherein the ratio of the mass of calcium ions to the mass of sodium ions in the culture medium is 15.3% by mass or less.

[0144] The method described in any of

[17]

[11] to

[16] , wherein the recovery step further comprises adding a chelating agent or a proteolytic enzyme to the culture medium.

[0145] The method described in any one of

[18]

[10] to

[17] , wherein the mass ratio of calcium ions to sodium ions in the swelling solution is 2.8% by mass or more and 48.5% by mass or less.

[0146]

[19]

[18] The method described herein, wherein the mass ratio of calcium ions to sodium ions in the swelling solution is 2.8% by mass or more and 15.3% by mass or less.

[0147]

[20] A pharmaceutical composition containing cells, comprising cells obtained by any of the methods described in [1] to

[19] .

[0148]

[21] A method for preparing a pharmaceutical composition containing cells, comprising the step of mixing cells obtained by any of the methods described in [1] to

[19] with a pharmaceutically acceptable excipient, solvent or cryopreservation solution.

Claims

1. Methods for culturing cells, It includes a culture step of culturing said cells in a culture medium using a cell culture carrier containing polysaccharides cross-linked by divalent or higher cations. The ratio of calcium ion mass to sodium ion mass in the culture medium is greater than 2.8% by mass and less than 48.5% by mass.

2. The method of claim 1, wherein, The cell culture carrier is a cell culture microcarrier.

3. The method of claim 1, wherein, The polysaccharides include alginic acid.

4. The method of claim 1, wherein, The culture step is performed using cell culture microcarriers containing alginate cross-linked with divalent or higher cations.

5. The method of claim 1, wherein, The divalent or higher cations include one or more selected from calcium ions, magnesium ions, divalent iron ions, trivalent iron ions, divalent silver ions, trivalent silver ions, copper ions, zinc ions, selenium ions, and other essential trace element ions.

6. The method of claim 4, wherein, The alginic acid cross-linked by divalent or higher cations includes calcium alginate.

7. The method of claim 1, wherein, The mass concentration of sodium ions in the culture medium is 2600–3600 ppm.

8. The method of claim 1, wherein, The cell culture carrier further includes a cell adhesion substance.

9. The method of claim 8, wherein, The cell adhesion substance includes one or more selected from gelatin, collagen, laminin, elastin, proteoglycan, fibronectin, fibronectin, peptides and domains having their activities, and serum.

10. The method of claim 1, further comprising a swelling step prior to the culture step, preparing the cell culture carrier by contacting and swelling the dried cell culture carrier with a swelling solution.

11. The method of claim 1, further comprising a recovery step of recovering the cultured cells after the culture step. The recovery step includes adjusting the ratio of calcium ion mass to sodium ion mass in the culture medium to less than 2.8 by mass.

12. The method of claim 1, wherein, The ratio of the mass of calcium ions to the sum of the masses of sodium ions and potassium ions in the culture medium is greater than 2.9% by mass and less than 168.8% by mass.

13. The method of claim 1, wherein, At the start of the culture step, the ratio of calcium ion mass to sodium ion mass in the culture medium is adjusted to be above 2.8% by mass and below 48.5% by mass.

14. The method of claim 1, wherein, Compared to the ratio of calcium ion mass to sodium ion mass in the culture medium at the beginning of the culture step, the ratio of calcium ion mass to sodium ion mass in the culture medium one day after the start of the culture step is more than 1.

15. The method of claim 1, wherein, The average particle size of the cell culture carrier is more than double that of the average particle size of the cell culture carrier one day after the start of the culture step, relative to the average particle size of the cell culture carrier at the beginning of the culture step.

16. The method of claim 1, wherein, The ratio of calcium ion mass to sodium ion mass in the culture medium is less than 15.3% by mass.

17. The method of claim 11, wherein, The recovery step further includes adding a chelating agent or a proteolytic enzyme to the culture medium.

18. The method of claim 10, wherein, The mass ratio of calcium ions to sodium ions in the swelling solution is greater than 2.8% by mass and less than 48.5% by mass.

19. The method of claim 18, wherein, The mass ratio of calcium ions to sodium ions in the swelling solution is greater than 2.8% by mass and less than 15.3% by mass.

20. A pharmaceutical composition containing cells, comprising cells obtained by the method of claim 1.

21. A method for preparing a pharmaceutical composition containing cells, comprising the step of mixing cells obtained by the method of claim 1 with a pharmaceutically acceptable excipient, solvent or cryopreservation solution.

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