Dry microcarriers and methods for producing the same

Abstract

To provide a method of producing a dry microcarrier for cell cultivation for obtaining a dry microcarrier with good cell adhesiveness and high storage stability.SOLUTION: A method for producing a dry microcarrier for cell cultivation is provided which comprises: a step of immersing, in a solvent including gelatin, an alginic acid fine particulate gel including cations having a valency of two or more; and a step of drying the alginic acid fine particulate gel immersed in the solvent, and forming a dry microcarrier. The dry microcarrier for cell cultivation comprises gelatin and an alginic acid cross-linked by a cation having a valency of two or more such that the gelatin is included inside the dry microcarrier for cell cultivation.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to dry microcarriers and a method for producing the same.

Background Art

[0002] In the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, etc., it is required to efficiently culture cells and tissues in large quantities. As a technique for culturing cells and the like in large quantities, the microcarrier culture method is known. The microcarrier culture method is, for example, a method in which cells, a culture solution, and microcarriers serving as an adhesion scaffold for the cells are introduced into a culture vessel, the culture solution is intermittently stirred to suspend the cells and the microcarriers, and the suspended cells contact the microcarriers while descending, thereby adhering to the surface of the microcarriers and growing (see, for example, Patent Document 1). Microcarriers can provide a very large adhesion and growth surface area with respect to the volume ratio, which is advantageous for large-scale cell culture.

[0003] So far, a method for producing a cell culture article including a step of forming microcarriers from a microcarrier composition containing a polygalacturonic acid compound or an alginic acid compound and a step of forming dry microcarriers has been known (see, for example, Patent Document 2), but the components constituting the dry microcarriers have low cell adhesiveness, and the surface of the microcarriers has been separately coated with a cell adhesive substance.

[0004] In addition, a cell culture carrier characterized by providing a coating layer made of collagen on the surface of beads made of granular alginate having a diameter of 1 to 5 mm is known (see, for example, Patent Document 3), but the inside of the cell culture carrier is not disclosed, and also, drying of the cell culture carrier is not known.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

[0006] There is a need to obtain a dry microcarrier that has good cell adhesion and high storage stability. [Means for solving the problem]

[0007] The present inventors have found a method for producing a dried microcarrier for cell culture, comprising the steps of immersing an alginate microparticle gel containing divalent or higher cations in a solvent containing gelatin, and drying the alginate microparticle gel immersed in the solvent to form a dried microcarrier, wherein the dried microcarrier contains gelatin and alginate crosslinked with divalent or higher cations, and the microcarrier produced by the production method containing gelatin internally has good cell adhesion and can be maintained in a dried state with high storage stability. This disclosure is based on these findings.

[0008] In other words, according to one aspect of this disclosure, A step of immersing alginate microparticle gel containing divalent or greater cations in a gelatin-containing solvent, and The process involves drying the alginate microparticle gel immersed in the aforementioned solvent to form dried microcarriers. A method for producing dry microcarriers for cell culture, including, The dry microcarrier for cell culture contains gelatin and alginic acid crosslinked with a divalent or higher cation, and contains gelatin inside the dry microcarrier for cell culture. A manufacturing method is provided.

[0009] According to another aspect of this disclosure, a dry microcarrier for cell culture, manufactured by the above-described manufacturing method, is provided.

[0010] According to another aspect of the present disclosure, a dry microcarrier for cell culture is provided, comprising gelatin and alginic acid crosslinked with divalent or greater cations, and containing gelatin internally. [Effects of the Invention]

[0011] The dried microcarriers for cell culture produced by the manufacturing method of this disclosure are advantageous in that they have good cell adhesion and high storage stability. [Brief explanation of the drawing]

[0012] [Figure 1] This image shows a phase-contrast microscope image of alginate microparticle gel. [Figure 2] This image shows a phase-contrast microscope image of a dried microcarrier manufactured by the manufacturing method disclosed herein. [Figure 3] This image shows a confocal laser microscope image of a dried microcarrier produced by the manufacturing method of this disclosure, near the center in the Z-axis direction. [Figure 4] This image shows a phase-contrast image of cells cultured one week after using dried microcarriers produced by the manufacturing method disclosed herein. [Figure 5] This image shows a fluorescence microscope image of cells stained with calcein after one week of cell culture using dried microcarriers produced by the manufacturing method disclosed herein. [Figure 6] This image shows a phase-contrast microscope image of cells treated with 10 mM EDTA one week after cell culture using dried microcarriers produced by the manufacturing method of this disclosure. [Figure 7] The images show phase-contrast microscope images (left) and fluorescence microscope images (right) of cells stained with calcein, at different gelatin concentrations after culturing. [Modes for carrying out the invention]

[0013] <Method for Producing Dry Microcarriers for Cell Culture> According to the present disclosure, there is provided a method for producing dry microcarriers for cell culture (hereinafter also referred to as "the dry microcarriers of the present disclosure" or simply "dry microcarriers" in this specification), comprising: a step of immersing alginate microparticle gel containing a divalent or higher cation in a solvent containing gelatin, and a step of drying the alginate microparticle gel immersed in the solvent to form dry microcarriers and the dry microcarriers for cell culture contain gelatin and alginate crosslinked with a divalent or higher cation, and contain gelatin inside the dry microcarriers for cell culture, a production method (also referred to as "the production method of the present disclosure" in this specification) is provided.

[0014] In the present disclosure, the "bonding" between the components constituting the alginate microparticle gel and the dry microcarriers for cell culture includes all forms of bonding, including both bonding with chemical bonds and bonding without chemical bonds. The bonding between the components constituting the dry microcarriers for cell culture may include one type of bonding form or may include two or more different types of bonding forms. Examples of the bonding between the components constituting the above-mentioned alginate microparticle gel and the dry microcarriers for cell culture include, for example, bonding between alginates, bonding between gelatins, bonding between alginate and gelatin, etc.

[0015] "Crosslinking" is a form of "bonding". In the present disclosure, expressions such as "crosslinked", "bonded by crosslinking", and similar expressions can be used interchangeably, all referring to the reversible or irreversible bonding of a plurality of the same or different molecules, either through other substances or without intervening substances. For example, "alginate crosslinked by a divalent or higher cation" refers to the reversible or irreversible bonding of a plurality of alginate molecules through a divalent or higher cation. In the present disclosure, unless otherwise defined, "chemical crosslinking" and similar expressions all refer to the irreversible bonding of a plurality of molecules through covalent bonds, and "physical crosslinking" and similar expressions all refer to the reversible bonding of a plurality of molecules through bonds that are not covalent bonds. Furthermore, "physical crosslinking" may or may not involve chemical bonds. Examples of physical crosslinking involving chemical bonds include the bonding of a plurality of molecules through chemical bonds (intermolecular forces) such as electrostatic attraction (ionic interaction) between molecules, hydrogen bonds, dipole-dipole interactions, and van der Waals forces. Examples of physical crosslinking not involving chemical bonds include mechanical (mechanical) adhesion and entanglement between molecules.

[0016] Alginate microparticle gel The alginate microparticle gel containing a divalent or higher cation used in the production method of the present disclosure can form an alginate microparticle gel by reacting droplets containing alginate with an aqueous solution containing a divalent or higher cation, and crosslinking the alginate with a divalent or higher cation. Note that the crosslinking of alginate by a divalent or higher cation is a reversible crosslinking (physical crosslinking involving chemical bonds) based on the electrostatic attraction between the anionic part (for example, carboxylic acid group (carboxyl group)) of the alginate molecule and the divalent or higher cation.

[0017] In the microcarriers of this disclosure, alginate is crosslinked by divalent or higher cations. Therefore, when using the microcarriers of this disclosure, the alginate can be decrosslinked by using a substance that competes with the divalent or higher cations contributing to the crosslinking and inhibits the crosslinking, thereby dissolving the microcarriers. Examples of such substances that inhibit crosslinking include chelating agents and monovalent cations. These substances that inhibit crosslinking may be used individually or in combination of two or more.

[0018] As a chelating agent, the same chelating agents described above for microcarriers can be used. The type of chelating agent is not particularly limited, and examples include ethylenediaminetetraacetic acid (EDTA), glycol etherdiaminetetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), HEDTA (N'-(2-hydroxyethyl)ethylenediamine-N,N,N'-triacetic acid), and nitrilotriacetic acid (NTA), which are commonly used in cell culture. Of the above-mentioned chelating agents, the use of EDTA is particularly preferred. Furthermore, the concentration of the chelating agent is not particularly limited and can be appropriately set depending on the type of chelating agent, the type of solvent in which the chelating agent is dissolved, etc. The concentration of the chelating agent can be, for example, 0.5 to 20 mM, 0.75 to 15 mM, 1 to 10 mM, etc.

[0019] The type of monovalent cation is not particularly limited and examples include sodium ions and potassium ions. The monovalent cation may be used dissolved in a solvent that does not contain monovalent cations, or dissolved in a solvent that does contain monovalent cations. Alternatively, the monovalent cation may be used in the form of a buffer solution containing monovalent cations (e.g., PBS). The concentration of the monovalent cation is not particularly limited and can be set appropriately depending on the type of monovalent cation, the type of solvent in which the monovalent cation is dissolved, etc. Examples of monovalent cation concentrations include 10-500 mM, 30-300 mM, and 50-200 mM.

[0020] Alginic acid droplets can be formed by extruding alginic acid into air or oil through a narrow nozzle, and then mixing them with an aqueous solution containing divalent or higher cations to form gelled microparticles. For example, by forming droplets of sodium alginate and mixing them with an aqueous solution of calcium chloride, alginic acid microparticle gel crosslinked with calcium alginate can be formed.

[0021] The fact that the alginic acid constituting the alginic acid nanoparticles is bonded by crosslinking with divalent or higher cations, as described above, can be confirmed by a method in which it is observed that when water is added to a dry microcarrier, gel-like particles are formed, and then when EDTA is added, these gel-like particles dissolve.

[0022] Alginic acid nanoparticles may contain alginic acid bonded by physical crosslinking in addition to alginic acid bonded by crosslinking with divalent or higher cations as described above. The physical crosslinking for forming such alginic acid may involve chemical bonding or not, but physical crosslinking without chemical bonding (e.g., adhesion or entanglement of alginic acid molecules) is preferred. Therefore, in one embodiment, the alginic acid nanoparticles contain alginic acid bonded by crosslinking with divalent or higher cations and alginic acid bonded by physical crosslinking.

[0023] Furthermore, even gels using general polysaccharides instead of the alginic acid mentioned above can be suitably dried and are expected to enhance cell adhesion. In particular, polysaccharides such as galacturonic acid, carrageenan, duran gum, and pectin, which gel with divalent or higher cations, can be suitably dissolved and applied to the manufacturing method disclosed herein. Although not bound by theory, polysaccharides, like alginic acid, have two or more carboxylic acid groups (carboxyl groups) and can suitably form cross-linked structures with divalent or higher cations, and are therefore considered to be usable for the formation of dried microcarriers.

[0024] Furthermore, when dispersing the alginate microparticle gel in a solution during the production of the dried microcarriers of this disclosure, it is preferable to include divalent or higher cations in the solution in order to suitably maintain the crosslinking structure between the alginate molecules constituting the alginate microparticle gel.

[0025] Divalent or greater cations The divalent or higher cations contained in the alginate microparticle gel are not particularly limited, but are preferably one or more selected from the group consisting of calcium ions, magnesium ions, ferrous ions, ferric ions, ferrous ions, ferrous ions, silver ions, silver ions, copper ions, zinc ions, selenium ions, and other essential trace element ions for humans (manganese ions, chromium ions, cobalt ions, etc.), and more preferably calcium ions.

[0026] gelatin The gelatin used in the manufacturing method of this disclosure is a substance that has high cell adhesion, is easy to dissolve, and is inexpensive. The gelatin may or may not be crosslinked. However, if the gelatin is irreversibly crosslinked, such as chemically crosslinked, the microcarriers become difficult to dissolve with chelating agents after cell culture, so it is preferable that the gelatin used in the manufacturing method of this disclosure is not irreversibly crosslinked, such as chemically crosslinked. Furthermore, it is preferable that the gelatin used in the manufacturing method of this disclosure conforms to the specifications for gelatin, purified gelatin, sterility, microbial limit, purity, or viscosity of the Japanese Pharmacopoeia, and it is more preferable that it conforms to the specifications for gelatin or purified gelatin of the Japanese Pharmacopoeia.

[0027] On the other hand, cross-linked gelatin has higher adsorption to alginate microparticle gel, which is preferable for cell culture. Therefore, from this viewpoint, it is preferable that gelatin be cross-linked, and from the viewpoint of the solubility of the microcarriers mentioned above, it is particularly preferable that reversible cross-linking such as physical cross-linking is performed. Physical cross-linking may involve chemical bonding or not. In addition to such physical cross-linking, gelatin may also include chemical cross-linking as described above. Here, it is preferable to adjust the cross-linking conditions in gelatin so that the cross-linking is weak enough to allow the microcarriers to dissolve easily after culture. Methods for cross-linking gelatin can be conventionally known methods for cross-linking molecules, and specifically include methods such as thermal cross-linking, ultraviolet cross-linking, and radiation cross-linking. In addition to gelatin, other cell adhesion substances may include scaffold proteins such as collagen, laminin, elastin, proteoglycans, fibronectin, and vitronectin, peptides or domains of sequences having their activity (e.g., RGD peptide, laminin E8, etc.), or serum. In that case, it is preferable that the chelating agent or the like is not irreversibly crosslinked and is contained within the microparticles so as not to interfere with the dissolution of the microparticles by the chelating agent after culturing.

[0028] The gelatin concentration in the solvent into which the alginate microparticle gel is immersed is not particularly limited, but a higher gelatin concentration is preferable because it results in better swelling and better cell adhesion and proliferation. The gelatin concentration in the solvent into which the alginate microparticle gel is immersed is preferably greater than 0.1% by mass, more preferably 0.5% by mass or more, even more preferably 1% by mass or more, even more preferably 2% by mass or more, and particularly preferably 4% by mass or more. When 1% by mass of alginate is used in the microparticle gel, the gelatin concentration can be increased up to a maximum of 99% by mass, but since the viscosity of the gelatin solution increases with increasing concentration, from the viewpoint of ease of handling the solution, the gelatin concentration in the solvent into which the alginate microparticle gel is immersed is preferably 20% by mass or less, more preferably 15% by mass or less, and even more preferably 10% by mass or less.

[0029] Because gelatin is contained within the alginic acid microparticle gel, the alginic acid microparticle gel can be swelled well by adding water again after drying. This is because the presence of gelatin between alginic acid molecules makes it difficult for new crosslinks to form between alginic acid molecules during drying. When preparing dried microcarriers, if the gelatin is positively charged, the negatively charged alginic acid can form poorly soluble complexes that are difficult to dissolve. Therefore, it is preferable to prepare dried microcarriers with a negatively charged gelatin. To achieve this, it is preferable to use gelatin with an acidic isoelectric point, such as alkali-treated gelatin, because the gelatin is negatively charged in a neutral state. Alternatively, when using gelatin with an alkaline isoelectric point, such as acid-treated gelatin, the gelatin is positively charged in a neutral state. Therefore, it is preferable to prepare dried microcarriers while making the gelatin negatively charged by increasing the hydroxide ion concentration of the solution used during the preparation of the dried microcarriers. Here, since the isoelectric point of acid-treated gelatin is 7 to 9, a pH of 9 or higher is preferable, and a pH of 9 to 13 is particularly preferable. Furthermore, when using cell adhesion substances other than gelatin, it is preferable to prepare the dried microcarriers in a negatively charged state. In addition, from the viewpoint of cell adhesion, it is more preferable that a portion of the gelatin is exposed on the surface of the microparticle gel.

[0030] The manufacturing method of the present disclosure includes the step of immersing an alginate microparticle gel containing divalent or higher cations in a solvent containing gelatin. The solvent containing gelatin is not particularly limited, but water is more preferred.

[0031] Furthermore, the manufacturing method of this disclosure includes a step of drying an alginate microparticle gel immersed in a gelatin-containing solvent to form dried microcarriers. By drying the microcarriers, the volume of each alginate microparticle decreases and they condense, causing the alginate constituting each alginate microparticle to adhere closely to a cell adhesion substance such as gelatin. As a result, physical crosslinks are formed between the alginate and the cell adhesion substance, and the two are strongly bonded together, which is thought to improve the cell adhesion of the microcarriers after drying (i.e., dried microcarriers).

[0032] Furthermore, the dried microcarriers of this disclosure have the advantages of high storage stability and ease of sterilization due to their dry state from which moisture has been removed. Generally, when microcarriers contain a relatively large amount of liquid components (i.e., when they are in a swollen state), chemical reactions (e.g., hydrolysis) occur between the liquid components and the components constituting the microcarriers, which can result in the decomposition of the microcarriers. On the other hand, when microcarriers contain substantially no liquid components (i.e., they are in a dry state), chemical reactions between the liquid components and the components constituting the microcarriers can be suppressed, and consequently, the decomposition of the microcarriers can be suppressed. Also, when microcarriers contain a relatively large amount of liquid components, if microorganisms such as bacteria become contaminated in the microcarriers, these microorganisms may proliferate through such liquid components. Since the dried microcarriers of this disclosure are used for cell culture, if microorganisms proliferate in the dried microcarriers, there is a possibility that a large amount of microorganisms will contaminate the target cells, and consequently, it may not be possible to sufficiently kill or suppress the growth of these microorganisms by using common antibiotics during cell culture. On the other hand, because the dry microcarriers are substantially free of liquid components, the proliferation of microorganisms via liquid components can be suppressed, and consequently, the contamination of target cells with microorganisms can be prevented. Furthermore, these microorganisms can be sufficiently killed or their growth inhibited by the use of common antibiotics during cell culture.

[0033] When forming dried microcarriers after immersing alginate microparticle gel in a gelatin-containing solvent, it is preferable to replace the gelatin-containing solvent (preferably water) on the outside of the alginate microparticle gel with a poor solvent for gelatin before the drying process, thereby surrounding the gelatin-impregnated alginate microparticle gel with the poor solvent for gelatin before drying. This is preferable because it removes most of the unwanted gelatin on the outside of the alginate microparticle gel, preventing the microcarriers from aggregating after drying, and also allows the gelatin to be trapped inside the microcarriers during drying. Specifically, for example, the alginate microparticle gel may be immersed in an aqueous gelatin solution to impregnate the alginate microparticle gel with gelatin, and then the gelatin on the outside of the alginate microparticle gel may be aggregated (molten into nanoparticles) with ethanol (a poor solvent for gelatin) and washed to remove the external gelatin, thereby surrounding the alginate microparticle gel with ethanol before drying.

[0034] According to a preferred embodiment of the manufacturing method of this disclosure, A step of immersing alginate microparticle gel containing divalent or higher cations in a gelatin-containing solvent. The process involves immersing the alginate microparticle gel, which has been immersed in the aforementioned gelatin-containing solvent, in a poor solvent for gelatin, and The process involves drying the alginate microparticle gel immersed in the aforementioned poor solvent to form dried microcarriers. A method for producing dry microcarriers for cell culture, including, The dry microcarrier for cell culture contains gelatin and alginic acid crosslinked with a divalent or higher cation, and contains gelatin inside the dry microcarrier for cell culture. A manufacturing method is provided.

[0035] Here, when replacing the solvent (preferably water) containing gelatin on the outside of the alginate microparticle gel with a poor solvent for gelatin before the drying process, it is preferable to perform the work in a way that minimizes shear stress on the alginate microparticle gel due to the poor solvent, so as not to remove the gelatin on the surface of the alginate microparticle gel. As the poor solvent, volatile solvents such as ethanol or isopropyl alcohol are preferred because they are easy to remove later.

[0036] Furthermore, by dropping a mixed droplet of alginic acid and gelatin into a poor solvent of gelatin containing divalent or higher cations, the gelatin-impregnated alginic acid microparticle gel can be surrounded by the poor solvent of gelatin. This method is preferable because it eliminates the need to replace, for example, water on the outside of the alginic acid microparticle gel with the poor solvent of gelatin.

[0037] Furthermore, the process of drying the alginic acid microparticle gel immersed in a gelatin-containing solvent is preferably carried out by vacuum drying, freeze-drying, dry heat drying, or a combination of these drying methods. Vacuum drying is preferably carried out for a drying time of 30 minutes or more. Dry heat drying is preferably carried out at a drying temperature of 100 to 200°C, more preferably 120 to 160°C, and for a time of 0.5 to 6 hours. The higher the drying temperature and the longer the drying time, the stronger the bonding of gelatin to gelatin in the microcarrier can be achieved through physical bonding such as mechanical adhesion and adsorption (physical crosslinking), and chemical bonding between alginic acid and gelatin due to a reaction called the Maillard reaction (chemical crosslinking). However, if the temperature is too high or the time is too long, it becomes difficult to dissolve the microcarrier with chelating agents or proteolytic enzymes, so this is undesirable.

[0038] It is more preferable that the gelatin is evenly dispersed within the dry microcarrier of this disclosure (i.e., the dry microcarrier for cell culture). Here, "evenly dispersed within" does not require that the gelatin be uniformly dispersed within the dry microcarrier, but rather means that as long as the gelatin is evenly dispersed within the dry microcarrier, some degree of uneven distribution of gelatin within it is acceptable. Furthermore, it is preferable that the amount of gelatin in the dry microcarrier of this disclosure be greater, and it is even more preferable that the amount of gelatin is greater than the amount of alginic acid in the dry microcarrier. In other words, it is preferable that the amount of gelatin in the dry microcarrier of this disclosure is greater than the amount of alginic acid. It is predicted that the gelatin will interpose between the alginic acid molecules and suppress the new crosslinking of the alginic acid molecules with divalent or higher cations during drying, thereby making it easier to swell to the shape before drying and suppressing the aggregation of microcarriers after drying.

[0039] The shape of the dried microcarriers of this disclosure is not particularly limited, but is preferably spherical, ellipsoidal, rod-shaped, sheet-shaped, polygonal, etc. The dried microcarriers of this disclosure may be sterilized after drying by a predetermined sterilization method. Examples of predetermined sterilization methods include dry heat sterilization, gamma ray sterilization, electron beam sterilization, and EOG (ethylene oxide gas) sterilization.

[0040] Dry microcarriers The dried microcarriers of this disclosure contain gelatin. Methods for confirming the presence of gelatin within the dried microcarriers include X-ray photoelectron spectroscopy (XPS), CHN elemental analysis, or reduced-pressure chemiluminescence, or a combination of two or more of these. That is, confirmation of whether or not the dried microcarriers of this disclosure contain gelatin is made by one of XPS, CHN elemental analysis, or reduced-pressure chemiluminescence, or by a combination of two or three of these methods. Of these, CHN elemental analysis and reduced-pressure chemiluminescence are preferably performed individually, or in combination with XPS and either CHN elemental analysis or reduced-pressure chemiluminescence. Specific methods for XPS, CHN elemental analysis, and reduced-pressure chemiluminescence are described below and in the subsequent examples.

[0041] In XPS, the dried microcarrier is ground, and measurements are taken before and after grinding ("before grinding" refers to the state before grinding, and "after grinding" refers to the state after grinding; the same applies hereinafter), and the change in N 1s / C 1s (atomic % / atomic %) is confirmed. For example, if N 1s / C 1s (atomic % / atomic %) before grinding is denoted as α(before grinding) and N 1s / C 1s (atomic % / atomic %) after grinding is denoted as α(after grinding), then a value of α(after grinding) / α(before grinding) of 0.08 or higher is preferable because it indicates that the dried microcarrier of this disclosure contains gelatin. Furthermore, considering the state after swelling, which will be described later, the value of α(after grinding) / α(before grinding) is preferably 0.1 or higher, more preferably 0.3 or higher, even more preferably 0.4 or higher, and particularly preferably 0.43 or higher. When the value of α(after grinding) / α(before grinding) is within the above range, the dried microcarriers can swell sufficiently, for example, the size of the swollen microcarriers can swell to a difference of 50% or less from the size before drying. Furthermore, considering the cell adhesion and proliferation properties of the swollen microcarriers, the value of α(after grinding) / α(before grinding) is preferably 0.45 or higher, more preferably 0.5 or higher, even more preferably 0.6 or higher, and particularly preferably 0.69 or higher. When the value of α(after grinding) / α(before grinding) is within the above range, sufficient cell adhesion and cell proliferation can be achieved. The pulverized dried microcarriers are powder obtained by grinding the dried microcarriers, and it is preferable that they are ground in a mortar until they are less than half of their original size.

[0042] Furthermore, the presence of gelatin inside the microcarriers can also be confirmed by combining the above-mentioned XPS with CHN elemental analysis or reduced-pressure chemiluminescence. When combining XPS with CHN elemental analysis or reduced-pressure chemiluminescence, if α is the N 1s / C 1s (atomic % / atomic %) obtained by XPS and β is the nitrogen content (mass%) obtained by CHN elemental analysis or reduced-pressure chemiluminescence, a β / α of 4.96 or higher is preferable because it indicates that gelatin is present inside the dried microcarriers. Moreover, considering the state after swelling, which will be described later, the value of β / α is preferably 10 or higher, more preferably 15 or higher, even more preferably 20 or higher, and particularly preferably 27 or higher. When the value of β / α is within the above range, the dried microcarriers can swell sufficiently, for example, the size of the swollen microcarriers can swell to a difference of 50% or less from the size before drying. Moreover, considering the cell adhesion and proliferation properties of the swollen microcarriers, the value of β / α is preferably 30 or higher, more preferably 35 or higher, even more preferably 40 or higher, and particularly preferably 44 or higher. By having a β / α value within the above range, sufficient cell adhesion and cell proliferation can be achieved. In this specification, the nitrogen content (mass%) β value is measured in the unground state of the dried microcarrier (before grinding).

[0043] Furthermore, using CHN elemental analysis and reduced-pressure chemiluminescence, the nitrogen content (Y) of the dry microcarrier is measured using the nitrogen composition ratio as the nitrogen content (mass%). Based on the nitrogen content of alginate alone (Z) and the nitrogen content of gelatin alone (X), the amount of gelatin (A) is calculated using the gelatin composition ratio of the dry microcarrier as the gelatin content (mass%).

[0044] Specifically, it is calculated using the formula A = (YZ) / (XZ). Since alginic acid contains almost no nitrogen, the amount of gelatin increases almost proportionally as the nitrogen content of the dry microcarriers increases.

[0045] If it is difficult to perform elemental analysis on alginic acid alone or gelatin alone, the nitrogen content of gelatin alone (X) may be set to 16.2% by mass (measured value), and the nitrogen content of alginic acid alone (Z) may be set to 0.031% by mass (measured value).

[0046] Furthermore, the nitrogen content in the dried microcarriers of this disclosure is preferably 0.43% by mass or more, relative to the total amount of dried microcarriers. Moreover, considering the state after swelling, the nitrogen content is preferably 1% by mass or more, more preferably 2% by mass or more, even more preferably 3% by mass or more, and particularly preferably 3.9% by mass or more. With the nitrogen content within the above range, the dried microcarriers can swell sufficiently, for example, the size of the microcarriers after swelling can swell to a difference of 50% or less from the size before drying. Moreover, considering the cell adhesion and proliferation properties of the microcarriers after swelling, the nitrogen content is preferably 4.5% by mass or more, more preferably 5% by mass or more, even more preferably 6% by mass or more, and particularly preferably 6.7% by mass or more. With the nitrogen content within the above range, sufficient cell adhesion and cell proliferation can be achieved.

[0047] In one embodiment, the nitrogen content and / or gelatin content in the dry microcarrier is measured and / or calculated by either CHN elemental analysis or reduced-pressure chemiluminescence alone. In this case, if the nitrogen content to be measured is greater than 0.5% by mass, measurement is performed by CHN elemental analysis, and if the nitrogen content to be measured is 0.5% by mass or less, measurement is performed by reduced-pressure chemiluminescence.

[0048] In another embodiment, the α and β values ​​in the dry microcarrier are measured by XPS and CHN elemental analysis or reduced-pressure chemiluminescence, respectively. In this case, if the nitrogen content of the sample to be measured is greater than 0.5% by mass, the measurement is performed by CHN elemental analysis, and if the nitrogen content of the sample to be measured is 0.5% by mass or less, the measurement is performed by reduced-pressure chemiluminescence.

[0049] According to a preferred embodiment of the manufacturing method of this disclosure, A step of immersing alginate microparticle gel containing divalent or greater cations in a gelatin-containing solvent, and The process involves drying the alginate microparticle gel immersed in the aforementioned solvent to form dried microcarriers. A method for producing dry microcarriers for cell culture, including, The dry microcarrier for cell culture consists of gelatin and alginic acid crosslinked with a divalent or higher cation, and contains gelatin inside the dry microcarrier for cell culture. A manufacturing method is provided.

[0050] The dried microcarriers of this disclosure can be used as cell culture microcarriers by swelling them as needed. Specifically, the dried microcarriers of this disclosure can be swelled with water before cell culture, and then cells can be seeded and allowed to adhere and proliferate on the surface of the microcarriers. On the other hand, the cell culture microcarriers obtained from the dried microcarriers of this disclosure can be reacted with a chelating agent to remove divalent or higher cations that are crosslinked with alginate from the microcarriers, thereby dissolving the microcarriers and recovering only the cells.

[0051] By adding, for example, an aqueous solution to the dried microcarriers of this disclosure, the dried microcarriers can absorb moisture and swell. Preferably, the degree of swelling of the dried microcarriers is such that it approximates the size and shape of the alginate microparticle gel immersed in the solvent before drying. By swelling to an extent that approximates the size and shape of the alginate microparticle gel immersed in the solvent before drying, transparency is improved, cell observation becomes easier, and microcarrier aggregation is less likely to occur, making cell culture easier.

[0052] The difference between the size (average particle diameter) of the microcarriers after swelling (microcarriers swollen with water) and the size (average particle diameter) of the alginate microparticle gel immersed in water before drying is preferably 50% or less, more preferably 25% or less, and even more preferably 10% or less. The difference (%) between the size (size after swelling) of the microcarriers after swelling (size after swelling) and the size (size before drying) of the alginate microparticle gel immersed in water before drying (size before drying) can be calculated using the following formula.

number

[0053] Since the dried microcarriers of this disclosure are used as cell culture carriers, it is preferable that their size at the time of use (i.e., after swelling) is greater than 10 μm, which is the approximate size of a cell. Furthermore, if they are too large during suspension culture, the sedimentation speed is fast, making culture difficult, so it is preferable that their size be 1 mm or less. Therefore, the size of the dried microcarriers of this disclosure at the time of use (i.e., after swelling) is not particularly limited, but is preferably 1 mm or less, more preferably 0.6 mm or less, and even more preferably 0.4 mm or less. The dried microcarriers of this disclosure are in a dry state, but when used in cell culture, they are usually used in a swollen state, so their size at the time of use (i.e., after swelling) is larger than that in the dry state. Therefore, in order to keep the size at the time of use within the above range, it is preferable that the size of the dried microcarriers of this disclosure (i.e., before swelling) is greater than 4.5 μm, more preferably 520 μm or less, more preferably 310 μm or less, and even more preferably 210 μm or less.

[0054] The aqueous solution used to swell the dried microcarriers of this disclosure preferably contains a cell adhesion substance. This is because the cell adhesion substance can be adsorbed onto the dried microcarriers produced by the manufacturing method of this disclosure, thereby improving cell adhesion. Suitable cell adhesion substances to be included in the aqueous solution for swelling include, for example, scaffold proteins such as gelatin, collagen, laminin, elastin, proteoglycans, fibronectin, and vitronectin, peptides or domains of sequences having the activity of these proteins (e.g., RGD peptide, laminin E8, etc.), or serum.

[0055] According to a preferred embodiment of the manufacturing method of this disclosure, A step of immersing alginate microparticle gel containing divalent or higher cations in a gelatin-containing solvent. A step of drying the alginate microparticle gel immersed in the aforementioned solvent to form dried microcarriers, and The step of swelling the dry microcarriers with a solvent (preferably water). A method for producing microcarriers for cell culture, which includes: The cell culture microcarrier contains gelatin and alginic acid crosslinked with a divalent or higher cation, and contains gelatin inside the cell culture microcarrier. A manufacturing method is provided.

[0056] According to a more preferred embodiment of the manufacturing method of this disclosure, A step of immersing alginate microparticle gel containing divalent or higher cations in a gelatin-containing solvent. A step of immersing alginate microparticle gel, which has been immersed in the aforementioned gelatin-containing solvent, in a poor solvent for gelatin, A step of drying the alginate microparticle gel immersed in the aforementioned poor solvent to form dried microcarriers, and The step of swelling the dry microcarriers with a solvent (preferably water). A method for producing microcarriers for cell culture, which includes: The cell culture microcarrier contains gelatin and alginic acid crosslinked with a divalent or higher cation, and contains gelatin inside the cell culture microcarrier. A manufacturing method is provided.

[0057] According to another embodiment of the manufacturing method of this disclosure, A method for producing microcarriers for cell culture, comprising the step of immersing alginate microparticle gel containing divalent or higher cations in a gelatin-containing solvent to form microcarriers, The cell culture microcarrier contains gelatin and alginic acid crosslinked with a divalent or higher cation, and contains gelatin inside the cell culture microcarrier. A manufacturing method is provided.

[0058] According to another preferred embodiment of the manufacturing method of the present disclosure, A step of immersing alginate microparticle gel containing divalent or greater cations in a gelatin-containing solvent, and A method for producing microcarriers for cell culture, comprising the step of immersing an alginate microparticle gel, which has been immersed in a solvent containing gelatin, in a poor solvent for gelatin to form microcarriers, The cell culture microcarrier contains gelatin and alginic acid crosslinked with a divalent or higher cation, and contains gelatin inside the cell culture microcarrier. A manufacturing method is provided.

[0059] In any of the above embodiments, the distribution and amount of gelatin in the cell culture microcarrier can be confirmed using a method such as XPS after drying the cell culture microcarrier, as described in the examples below.

[0060] cell culture After swelling the dried microcarriers produced by the manufacturing method of this disclosure with the above aqueous solution, cells can be seeded and the cells can adhere to the surface of the microcarriers to allow for cell proliferation. Cell proliferation can be carried out under static conditions, or the microcarriers can be suspended and cultured by stirring the culture medium. Suspension culture is preferred in order to culture a larger number of cells using microcarriers.

[0061] Adherent cells are preferably used as cells that can be cultured on the surface of the microcarrier, and examples of such cells include hepatocytes (parenchymal cells of the liver), Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, epidermal cells such as fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells and epidermal keratinocytes, epithelial cells such as tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells and corneal epithelial cells, mammary gland cells, pericytes, myoblasts, myotubes, satellite cells, muscle cells such as smooth muscle cells and cardiomyocytes, renal cells, pancreatic islet cells, nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, and osteocytes. These cells may be primary cells directly collected from tissues or organs, or they may be passaged several times. Furthermore, these cells may be undifferentiated cells such as embryonic stem cells and iPS cells, somatic stem cells such as mesenchymal stem cells with differentiation potential, unipotent stem cells such as vascular endothelial progenitor cells with single differentiation potential, or cells that have completed differentiation. The cells used with the microcarrier may also include CHO cells, 293 cells, 3T3 cells, Vero cells, MRC5 cells, HeLa cells, hybridomas, and other cell lines derived from these, which are widely used as cell substrates for the production of biopharmaceuticals and viral vectors. Furthermore, the cells used for culture may be a single type of cell or two or more types of cells may be co-cultured. By culturing such cells using microcarriers, it is possible to manufacture medical products and food products that are expected to have therapeutic effects.

[0062] Dissolution To separate and recover cells from microcarriers after culture, the microcarriers can be easily separated and recovered by dissolving them. By reacting the microcarriers produced by the manufacturing method of this disclosure with a chelating agent, the divalent or higher cations crosslinking the alginate are removed from the alginate, and the microcarriers can be dissolved.

[0063] EDTA is preferably used as a chelating agent for dissolving microcarriers. Furthermore, adding alginate-degrading enzymes or proteolytic enzymes in addition to the chelating agent can hydrolyze alginic acid and gelatin, allowing for faster dissolution. On the other hand, from the viewpoint of minimizing damage to cells, it is preferable not to use proteolytic enzymes. Furthermore, from the viewpoint of safety management and cost, it is preferable not to use alginate-degrading enzymes to minimize the number of added substances. Therefore, it is most preferable that the microcarriers can be dissolved using only a chelating agent.

[0064] <Microcarriers for cell culture> Another aspect of the present disclosure provides a microcarrier for cell culture. In this specification, “microcarrier for cell culture” means both the microcarrier before the “drying” step in the manufacturing method of the present disclosure and the microcarrier after the dried microcarrier of the present disclosure (i.e., the dried microcarrier for cell culture) has been swollen as necessary (i.e., the microcarrier for cell culture).

[0065] According to another aspect of the present disclosure, a microcarrier for cell culture is provided, comprising gelatin and alginic acid crosslinked with a divalent or greater cation, and containing gelatin internally.

[0066] Furthermore, according to another preferred embodiment of this disclosure, a dry microcarrier for cell culture is provided, comprising gelatin and alginic acid crosslinked with a divalent or higher cation, and containing gelatin internally. Preferably, the gelatin inside the microcarrier is evenly dispersed within the microcarrier. Here, the dry microcarrier has a water content of 50% by mass or less, more preferably 30% by mass or less, even more preferably 25% by mass or less, even more preferably 20% by mass or less, even more preferably 15% by mass or less, even more preferably 10% by mass or less, even more preferably 5% by mass or less, even more preferably 2% by mass or less, and even more preferably 1% by mass or less, as measured by the atmospheric pressure heating drying method. The water content inside the microcarrier is measured by the atmospheric pressure heating drying method in accordance with the method described in the examples.

[0067] The gelatin, divalent or higher cations, alginic acid, etc. contained in the cell culture microcarrier may be the same as the gelatin, divalent or higher cations, alginic acid, etc. used in the method for producing the dried cell culture microcarrier described in this disclosure. [Examples]

[0068] The present disclosure will be described in more detail below with reference to examples, but the present disclosure is not limited to the following examples.

[0069] Test Example 1: Preparation of dried microcarriers, as well as their swelling, cell culture, and lysis. 1-1-1 <Dry microcarrier fabrication> A 1% by mass aqueous solution of sodium alginate (Fujifilm Corporation, 194-13321) and a 1% by mass aqueous solution of calcium chloride (Fujifilm Corporation, 038-24985) were prepared, respectively.

[0070] A sodium alginate aqueous solution was dropped into a calcium chloride aqueous solution using a 32G syringe needle to prepare calcium alginate microparticle gels with a diameter of approximately 200 μm (measured by optical microscope observation) (the alginate is crosslinked by calcium ions) (see Figure 1). The microparticle gels were washed with 70% ethanol and water, and the microparticle gels were recovered using a cell strainer (Falcon, 352340). The microparticle gels were immersed in a 4% by mass aqueous solution of autoclaved alkali-treated gelatin (Sigma-Aldrich, G9391-100G) and left at 20°C for more than 2 hours to allow the gelatin to permeate into the microparticle gels.

[0071] Here, the size of the microparticle gel remained approximately 200 μm in diameter, and no change in shape was observed. After collecting the microparticle gel with a cell strainer, the microparticle gel was immersed in ethanol and collected with the cell strainer to remove the gelatin from the outside of the microparticle gel and encapsulate the gelatin within the microparticle gel. Optical microscope observation confirmed that the calcium alginate microparticle gel before gelatin encapsulation was colorless, and the gelatin aqueous solution was slightly yellowish. However, after surrounding the microparticle gel with ethanol and encapsulating the gelatin, the inside of the microparticle gel was slightly yellowish, while the outside of the microparticle gel was colorless, thus confirming that the gelatin was encapsulated within the microparticle gel.

[0072] After vacuum drying in this state, dry heat drying at 150°C for 2 hours removed the water, reducing the size to approximately 90 μm (measured by optical microscopy), and a powdery dried microcarrier (dried microcarrier produced by the manufacturing method of this disclosure) was obtained (see Figure 2). At this point, the material was a white powder immediately after vacuum drying, but it changed to brown after dry heat drying.

[0073] 1-1-2 Furthermore, in the method for preparing the powdered dry microcarriers described above, dry microcarriers were prepared separately in the same manner as above, except that fluorescently stained gelatin (Thermo Fisher, G13187) was added to a 4% by mass aqueous solution of autoclaved alkali-treated gelatin (Sigma-Aldrich, G9391-100G) to a concentration of 0.01% by mass. When the prepared dry microcarriers were observed with a confocal laser microscope, it was confirmed that the entire interior was stained, confirming that the gelatin was uniformly dispersed throughout the dry microcarrier (see Figure 3: a pinhole was prepared so that the width of the slice image (cross-section of the dry microcarrier) was sufficiently smaller than the radius of the dry microcarrier). Similarly, in the microcarriers after swelling described in <Swelling, Cell Culture, and Dissolution> below, it was observed with a confocal laser microscope that the entire interior of the microcarrier was stained, confirming that the gelatin was uniformly dispersed throughout the dry microcarrier.

[0074] <Swelling, cell culture, and lysis> When 1 / 100th the volume of a 10% calcium chloride aqueous solution was added to MSCGM BulletKit® (Lonza Corporation, PT-3001) culture medium, the dried microcarriers prepared above were immersed in this medium and left at 20°C for 30 minutes. They swelled to a diameter of approximately 200 μm, becoming almost the same size as the calcium alginate microparticle gel before drying. Subsequently, human mesenchymal stem cells (manufacturer: Lonza Corporation, PT-2501) were seeded, and it was confirmed that the cells adhered to and grew on the surface of the microparticle gel. After one week of culture, it was confirmed that the cells proliferated to the point where they almost completely covered the surface of the microparticles (see Figures 4 and 5). After one week of culture, the medium was removed, a 10 mM EDTA solution was added, and after standing for about 1 minute, the microparticle gel dissolved by pipetting, and the separation of cells from the microparticle gel was confirmed by microscopy (see Figure 6).

[0075] 1-2 Dry microcarriers (including dry heat drying) similar to those described in 1-1-1 above were prepared separately, and two types were subjected to atmospheric pressure heating drying (measurement conditions: dryer temperature 150°C, drying time 2 hours): a sample immediately after preparation and a stored sample (storage conditions: 100% humidity, 25°C, 3 days). The moisture content of the sample immediately after preparation was 0% by mass, and the moisture content of the stored sample was 22.5% by mass. Furthermore, when the same method as described above was used to prepare both freshly prepared and stored samples, and the above-mentioned swelling, cell culture, and lysis tests were performed on both, the same results as in the above tests were observed, confirming that similar effects can be obtained in the water content range of 0 to 22.5% by mass.

[0076] 1-3 Microcarriers before dry heat drying were prepared separately using the same method as described in 1-1-1 above (obtained by immersing in ethanol and collecting with a cell strainer to remove gelatin from the outside of the microparticle gel and encapsulating the gelatin within the microparticle gel). These microcarriers were then immersed in water, and their size at that time was measured as the "size before drying (average particle diameter)," which was approximately 200 μm in diameter. In addition, dried microcarriers after dry heat drying were prepared separately using the same method as described in 1-1-1 above (obtained by performing dry heat drying), and their size after swelling in water at 20°C for 30 minutes was measured as the "size after swelling (average particle diameter)." Here, the size after swelling was approximately 200 μm in diameter, which is almost the same as the size after swelling using the culture medium described in <Swelling, cell culture, and lysis> of Test Example 1 above.

[0077] The method for measuring the average particle size is as follows: (Method for measuring average particle size) Dry microcarriers are placed in a transparent container with a flat bottom for observation, and the dry microcarriers are allowed to settle. Optical microscope observation is then performed, and the average of the equivalent spherical diameters of all observed dry microcarriers is defined as the average particle size. When placing the dry microcarriers into the observation container, the bottom area of ​​the container and the amount of dry microcarriers placed in it are adjusted so that at least 10 dry microcarriers can be observed without overlapping, and measurements are taken accordingly.

[0078] Furthermore, in the above-mentioned dried microcarriers, the difference (%) between the size after swelling and the size before drying was calculated to be 0% based on the following formula.

[0079]

number

[0080] Test Example 2: Preparation and evaluation of dried microcarriers at various gelatin concentrations. Except for the gelatin concentration in the gelatin aqueous solution into which the calcium alginate microparticle gel was immersed being 0% by mass, 0.1% by mass, 0.5% by mass, 1% by mass, or 2% by mass, dried microcarriers were prepared in the same manner as described in 1-1-1 of Test Example 1, and then swelled, cultured, and dissolved (see Figure 7).

[0081] Furthermore, for dried microcarriers prepared in the same manner as in Test Example 1, except that the gelatin concentration was set to 0% by mass, 0.1% by mass, 0.5% by mass, 1% by mass, or 2% by mass, the difference between the size of the microcarriers after swelling (size after swelling) and the size of the alginate microparticle gel immersed in water before drying (size before drying) was calculated using the above formula, in the same manner as in 1-3 above.

[0082] The results are shown in Table 1 below. Here, the size after swelling was approximately the same as the size after swelling when using the culture medium described in <Swelling, Cell Culture, and Lysis> of Test Example 1 above. [Table 1]

[0083] As shown in Table 1, when the gelatin concentration in the gelatin aqueous solution into which the calcium alginate microparticle gel was immersed was 0%, 0.1%, 0.5%, 1%, or 2%, the size of the microparticles after swelling of the dried microcarriers (measured by optical microscopy) became closer to the size before drying as the gelatin concentration increased, reaching approximately 60 μm, 70 μm, 120 μm, 150 μm, and 200 μm, respectively. This indicates that the presence of gelatin in the microparticles facilitates good swelling.

[0084] In this study, when the gelatin concentration in the gelatin aqueous solution into which the calcium alginate microparticle gel was immersed was 0% by mass and 0.1% by mass, aggregation of the microparticles was observed after the drying of the microcarriers, but almost no aggregation was observed in other cases. Regarding cell adhesion, when the gelatin concentration in the gelatin aqueous solution into which the calcium alginate microparticle gel was immersed was 0% by mass, almost no cell adhesion was observed. Regarding cell adhesion, when the gelatin concentration in the gelatin aqueous solution into which the calcium alginate microparticle gel was immersed was 0.1% by mass and 0.5% by mass, cell adhesion was observed, but the cells did not proliferate enough to cover the surface of the microparticles. Regarding cell adhesion, when the gelatin concentration in the gelatin aqueous solution into which the calcium alginate microparticle gel was immersed was 1% by mass and 2% by mass, cell adhesion was observed, and cell proliferation was confirmed to the extent that it covered the surface of the microparticles. From these results, it was found that when the gelatin concentration in the gelatin aqueous solution into which the calcium alginate microparticle gel was immersed was 0.5% by mass or higher, the size (average particle diameter) of the dried microcarriers after swelling was less than 50% of the size (average particle diameter) before drying, and swelling was good with almost no aggregation. Furthermore, it was found that when the gelatin concentration in the gelatin aqueous solution into which the calcium alginate microparticle gel was immersed was 0.1% by mass or higher, the cell adhesion and proliferation properties were significantly improved compared to when the gelatin concentration in the gelatin aqueous solution was 0% by mass. Furthermore, it was found that when the gelatin concentration in the gelatin aqueous solution into which the calcium alginate microparticle gel was immersed was 1% by mass or higher, swelling was good, and cell adhesion and proliferation were also good, with the microparticles almost completely covering the surface of the swollen microcarriers. Furthermore, in all cases where the gelatin concentration was 0%, 0.1%, 0.5%, 1%, and 2%, after culturing for one week, the culture medium was removed, a 10 mM EDTA solution was added, and after standing for about 1 minute, the microparticle gel dissolved by pipetting, and the separation of cells from the microparticle gel was confirmed by microscopy.

[0085] Furthermore, when the same evaluation as in Test Example 1 was performed for a gelatin concentration of 4% by mass, the size of the dried microcarriers after swelling was approximately 200 μm, indicating good swelling, cell adhesion, and cell proliferation to the point of covering the surface of the microparticles. In addition, under all the gelatin concentration conditions examined here, after one week of culture, the culture medium was removed, a 10 mM EDTA solution was added, and after standing for about 1 minute, the microparticle gel dissolved by pipetting, and the separation of cells from the microparticle gel was confirmed by microscopy.

[0086] Test Example 3: Examination of the amount of gelatin in dried microcarriers at various gelatin concentrations. Dry microcarriers (dry microcarriers manufactured by the manufacturing method of this disclosure) were prepared by the same method as described in 1-1-1 of Test Example 1 and Test Example 2 above, and the amount of gelatin (mass%) inside the dry microcarriers was measured by performing elemental analysis of these by CHN elemental analysis or reduced-pressure chemiluminescence. The specific procedures and measurement conditions for CHN elemental analysis and reduced-pressure chemiluminescence are as follows.

[0087] (CHN elemental analysis method) The sample was weighed to an accurate degree of 0.0001 mg, and simultaneous CHN analysis was performed. <Measurement conditions> System: vario MICRO cube (manufactured by Elementar) Combustion furnace: 1150℃ Reduction furnace: 850℃ Helium flow rate: 200 mL / min Oxygen flow rate: 25-30 mL / min

[0088] (Chemiluminescence under reduced pressure) The sample was weighed and introduced into the analyzer, where it was thermally decomposed and oxidized to produce nitric oxide, which was then measured by chemiluminescence. Quantitative analysis was performed automatically using a calibration curve prepared beforehand with pyridine standard solution. <Measurement conditions> Equipment: Trace nitrogen analyzer ND-100 (manufactured by Mitsubishi Chemical Corporation) Electric furnace temperature (horizontal reactor) Pyrolysis part: 800℃ Catalyst part: 900℃ O2 flow rate (main / sub): 300 ml / min (set value) / 300 ml / min (set value) Ar flow rate: 400 ml / min (set value) Sens.: Low

[0089] Here, for dried microcarriers prepared using only gelatin, and when the gelatin concentration in the gelatin aqueous solution in which the calcium alginate microparticle gel was immersed was 4% by mass, 2% by mass, 1% by mass, or 0.5% by mass, the nitrogen content (by mass) in the dried microcarriers was measured by CHN elemental analysis. For dried microcarriers prepared using the gelatin aqueous solution in which the calcium alginate microparticle gel was immersed was 0.1% by mass or 0% by mass (alginate only), the nitrogen content (by mass) in the dried microcarriers was measured by reduced-pressure chemiluminescence. The amount of gelatin (by mass) was calculated from the nitrogen content (by mass) of alginate alone and gelatin alone. Specifically, the nitrogen content (Y) of the dry microcarriers was measured using CHN elemental analysis or reduced-pressure chemiluminescence, with the nitrogen composition ratio defined as the nitrogen content (mass%). Based on the nitrogen content of alginate alone (Z) and the nitrogen content of gelatin alone (X), the gelatin content (A) was calculated using the gelatin composition ratio of the dry microcarriers, with the gelatin content (mass%) defined as the gelatin content (mass%), according to the following formula. A = (YZ) / (XZ) Table 2 below shows the nitrogen content and gelatin content of the dry microcarriers, as well as the swelling and culture results, for each gelatin concentration condition. When only gelatin was used, the nitrogen content in the dry microcarriers was 16.2% by mass. Furthermore, when the gelatin concentration in the gelatin aqueous solution into which the calcium alginate microparticle gel was immersed was 0.5% by mass, the nitrogen content in the dry microcarriers was 3.9% by mass, and the gelatin content was 24% by mass. From the results in Table 2 below, it was found that the higher the gelatin concentration in the gelatin aqueous solution into which the calcium alginate microparticle gel was immersed, the greater the nitrogen content and gelatin content in the dry microcarriers. The evaluation criteria for "swelling" and "cell adhesion and proliferation" are as follows. The evaluation methods for "swelling" and "cell adhesion and proliferation" were carried out according to the method described in Test Example 2.

[0090] (1) Swelling <Evaluation Criteria> ○: The size (average particle diameter) of the dried microcarriers after swelling is 50% or less of the size (average particle diameter) before drying. △: The size (average particle diameter) of the dried microcarriers after swelling is greater than 50% but less than 70% of the size (average particle diameter) before drying. ×: The size (average particle diameter) of the dried microcarriers after swelling is 70% or more of the size (average particle diameter) before drying.

[0091] (2) Cell adhesion and proliferation <Evaluation Criteria> ○: Cell adhesion is observed, and cell proliferation is confirmed to the extent that it covers the surface of the microparticles. △: Cell adhesion is observed, but the cells do not proliferate enough to cover the surface of the microparticles. ×: Almost no cell adhesion is observed.

[0092] <Amount of gelatin in dry microcarriers at each gelatin concentration> Dry microcarriers were prepared using the same method as described in Test Example 2, and the amount of gelatin on the surface of the dry microcarriers was measured as N 1s / C 1s (atomic % / atomic %) by XPS analysis. Here, N 1s is a value derived from nitrogen atoms from the gelatin, and C 1s is a value derived from carbon atoms in the entire dry microcarrier. Al Kα rays were used as the photoelectron excitation source for the XPS analysis, and the specific instrument used was the ESCA-3400 (KRATOS ANALYTICAL).

[0093] Table 3 shows the amount of gelatin on the surface of the dried microcarriers prepared at each gelatin concentration in the gelatin aqueous solution into which the calcium alginate microparticle gel was immersed, along with the nitrogen content, swelling, and culture results described in Table 2.

[0094] Table 2 also shows the β / α values, where α is defined as N 1s / C 1s (atomic % / atomic %) obtained by XPS analysis, and β is defined as the nitrogen content (mass%) obtained by CHN elemental analysis or reduced-pressure chemiluminescence. For dry microcarriers prepared with gelatin alone, and with gelatin concentrations of 4 mass%, 2 mass%, 1 mass%, and 0.5 mass% in the gelatin aqueous solution into which the calcium alginate microparticle gel was immersed, the nitrogen content (mass%) β was measured by CHN elemental analysis. For dry microcarriers prepared with gelatin concentrations of 0.1 mass% and 0 mass% (alginate only) in the gelatin aqueous solution into which the calcium alginate microparticle gel was immersed, the nitrogen content (mass%) β was measured by reduced-pressure chemiluminescence. Note that the above-mentioned α and β values ​​were measured with the dry microcarriers in an unground state (before pulverization).

[0095] Here, considering the characteristics (measurement thresholds) of CHN elemental analysis and reduced-pressure chemiluminescence, forms with a nitrogen content of 0.5 mass% or less were measured by reduced-pressure chemiluminescence, while forms with a nitrogen content exceeding 0.5 mass% were measured by CHN elemental analysis. Furthermore, the specific procedures and measurement conditions for CHN elemental analysis and reduced-pressure chemiluminescence were the same as those for CHN elemental analysis and reduced-pressure chemiluminescence in the aforementioned test example.

[0096] As shown in the results for α in Table 3 below, the amount of gelatin on the surface of the dried microcarrier hardly increases when the gelatin concentration in the gelatin aqueous solution into which the calcium alginate microparticle gel is immersed is 0.5% by mass or higher. On the other hand, the results in Table 3 below show that increasing the gelatin concentration in the gelatin aqueous solution into which the calcium alginate microparticle gel is immersed increases the nitrogen content (β) (gelatin amount) of the entire dry microcarrier, indicating that gelatin is supported inside the dry microcarrier in a manner dependent on the gelatin concentration.

[0097] Furthermore, as described above, α (amount of nitrogen on the surface of the dry microcarrier) hardly increased when the gelatin concentration in the gelatin aqueous solution into which the calcium alginate microparticle gel was immersed was 0.5% by mass or higher, and it was found that if β / α was 4.96 or higher, gelatin was supported inside the dry microcarrier. In addition, XPS analysis was performed on the dry microcarriers prepared by the same method as described in Test Example 2 above, before and after grinding, and α (N 1s / C 1s) before and after grinding was determined. Al Kα rays were used as the photoelectron excitation source for the XPS analysis, and the specific instrument used was the ESCA-3400 (manufactured by KRATOS ANALYTICAL). The values ​​of α (after grinding) / α (before grinding), where N 1s / C 1s (atomic % / atomic %) before grinding is defined as α (before grinding) and N 1s / C 1s (atomic % / atomic %) after grinding is defined as α (after grinding), are shown in Table 3 below. When α(after grinding) / α(before grinding) was 0.08 or higher, it was found that gelatin was supported inside the dried microcarrier.

[0098] [Table 2]

[0099] [Table 3]

[0100] Test Example 4: Storage Stability Test A storage stability test was conducted on dried microcarriers prepared with a gelatin concentration of 4% by mass using the same method as described in 1-1-1 of Test Example 1. Specifically, each prepared dried microcarrier was stored at room temperature for 18 months, and then each dried microcarrier was subjected to swelling, cell culture, and lysis according to the method described in 1-1 of Test Example 1. As a result, it was confirmed that the performance of each dried microcarrier after 18 months of storage at room temperature was comparable to that of each dried microcarrier before storage. This result indicates that the dried microcarriers of this disclosure have good storage stability.

[0101] Test Example 5: Microcarrier preparation, swelling, cell culture, and lysis thereof. In the preparation of the dried microcarriers in Test Example 1, the microcarriers were prepared using the same method as in Test Example 1, except that the gelatin infiltration into the microparticle gel was performed by immersing the microparticle gel in a 2% by mass gelatin aqueous solution for one day, and that a drying step was omitted. When mouse skeletal myoblasts were seeded onto the prepared microcarriers and cultured in DMEM medium, cell adhesion and proliferation on the microcarriers were confirmed.

[0102] In the above-described method for preparing microcarriers, each microcarrier was prepared in the same manner as described above, except that the 1% by mass aqueous solution of calcium chloride was replaced with a 1% by mass aqueous solution of magnesium chloride, a 1% by mass aqueous solution of iron(II) chloride, and a 1% by mass aqueous solution of iron(III) chloride, respectively. As described above, when mouse skeletal myoblasts were seeded onto the prepared microcarriers and cultured in DMEM medium, cell adhesion and proliferation were confirmed in all microcarriers.

[0103] When the cell adhesion and proliferation properties of each of the microcarriers described above were evaluated using the same method as described in Test Example 3, all results fell under the category of "△: Cell adhesion is observed, but cells do not proliferate enough to cover the surface of the microparticles."

[0104] In this test example, the microcarriers were not prepared using a drying process. However, it is believed that a drying process would improve cell adhesion and storage stability. Specifically, drying the microcarriers reduces the volume of each alginate microparticle, causing them to condense and allowing the alginate constituting each microparticle to adhere closely to cell adhesion materials such as gelatin. As a result, physical crosslinks are formed between the alginate and the cell adhesion material, leading to a strong bond and improved cell adhesion. Furthermore, drying the microcarriers results in a state that is substantially free of liquid components such as water. This suppresses chemical reactions such as hydrolysis caused by liquid components and the growth of microorganisms such as bacteria, thus improving storage stability. Therefore, given that each microcarrier exhibited good cell adhesion and high storage stability even without a drying process, it is highly probable that if these microcarriers undergo a drying process, they will exhibit cell adhesion and high storage stability similar to or even better than those without a drying process.

[0105] Another aspect of the present invention relates to the following [1] to

[18] . [1] A step of immersing alginate microparticle gel containing divalent or greater cations in a gelatin-containing solvent, and The process involves drying the alginate microparticle gel immersed in the aforementioned solvent to form dried microcarriers. A method for producing dry microcarriers for cell culture, including, The dry microcarrier for cell culture contains gelatin and alginic acid crosslinked with a divalent or higher cation, and contains gelatin inside the dry microcarrier for cell culture. Manufacturing method. [2] The manufacturing method according to [1], comprising the step of immersing the alginic acid microparticle gel, which has been immersed in the solvent containing gelatin, in a poor solvent for gelatin before drying. [3] The manufacturing method according to [2], wherein the poor solvent is a volatile solvent. [4] The manufacturing method according to any one of [1] to [3], wherein the dried microcarrier for cell culture is a salt of gelatin and alginic acid crosslinked with a divalent or higher cation. [5] The manufacturing method according to any one of [1] to [4], wherein the gelatin content in the solvent is more than 0.1% by mass relative to the total amount of the solvent. [6] The manufacturing method according to any one of [1] to [5], wherein the gelatin content of the entire dry microcarrier for cell culture is 2.5% by mass or more relative to the total amount of the dry microcarrier for cell culture. [7] The manufacturing method according to any one of [1] to [6], wherein the nitrogen content in the dry microcarrier for cell culture is 0.43% by mass or more relative to the total amount of the dry microcarrier for cell culture. [8] The method for producing a product according to any one of [1] to [7], wherein the divalent or greater cation is one or more selected from the group consisting of calcium ions, magnesium ions, ferrous ions, ferric ions, ferrous ions, ferrous ions, ferrous ions, copper ions, zinc ions, selenium ions, and other human essential trace element ions. [9] The manufacturing method according to any one of [1] to [8], wherein the gelatin contained inside the dried microcarrier for cell culture is bound to alginic acid. A dried microcarrier for cell culture, manufactured by the manufacturing method described in any of

[10] [1] to [9].

[11] A dry microcarrier for cell culture comprising gelatin and alginic acid crosslinked with divalent or higher cations, and containing gelatin internally.

[12] A dry microcarrier for cell culture comprising gelatin and alginic acid crosslinked with a divalent or higher cation, and containing gelatin internally, The average particle size of the aforementioned dried microcarriers for cell culture is 1 mm or less. Dry microcarriers for cell culture.

[13] The dry microcarrier for cell culture according to

[11] or

[12] , wherein the gelatin is evenly dispersed inside the dry microcarrier for cell culture.

[14] A dry microcarrier for cell culture comprising gelatin and alginic acid crosslinked with divalent or greater cations, Let α be the N 1s / C 1s (atomic % / atomic %) of the dried microcarrier for cell culture obtained by X-ray photoelectron spectroscopy (XPS). When the nitrogen content (mass%) of the dried microcarriers for cell culture obtained by CHN elemental analysis or reduced-pressure chemiluminescence is denoted as β, The ratio of β(before grinding) / α(before grinding) is 4.96 or higher, or The ratio of α(after grinding) / α(before grinding) is 0.08 or higher. (Here, "α (before grinding)" and "β (before grinding)" represent the N 1s / C 1s (atomic % / atomic %) and nitrogen content (mass %) of the dried microcarrier for cell culture, respectively, and "α (after grinding)" represents the N 1s / C 1s (atomic % / atomic %) of the powder obtained by grinding the dried microcarrier for cell culture.) Dry microcarriers for cell culture.

[15] The above β(before grinding) / α(before grinding) is 4.96 or more, and The dry microcarrier for cell culture according to

[14] , wherein the ratio of α(after grinding) / α(before grinding) is 0.08 or greater.

[16] A dry microcarrier for cell culture comprising gelatin and alginic acid crosslinked with divalent or higher cations, Let α be the N 1s / C 1s (atomic % / atomic %) of the dried microcarrier for cell culture obtained by X-ray photoelectron spectroscopy (XPS). When the nitrogen content (mass%) of the dried microcarriers for cell culture obtained by CHN elemental analysis or reduced-pressure chemiluminescence is denoted as β, β(before grinding) / α(before grinding) is 4.96 or higher, α(after grinding) / α(before grinding) is 0.08 or higher. (Here, "α (before grinding)" and "β (before grinding)" represent the N 1s / C 1s (atomic % / atomic %) and nitrogen content (mass %) of the dried microcarrier for cell culture, respectively, and "α (after grinding)" represents the N 1s / C 1s (atomic % / atomic %) of the powder obtained by grinding the dried microcarrier for cell culture.) The average particle size of the aforementioned dried microcarriers for cell culture is 1 mm or less. Dry microcarriers for cell culture.

[17] The dry microcarrier for cell culture according to any one of

[10] to

[16] , wherein the gelatin content of the entire dry microcarrier for cell culture is 2.5% by mass or more relative to the total amount of the dry microcarrier for cell culture.

[18] The dry microcarrier for cell culture according to any one of

[10] to

[17] , wherein the nitrogen content in the dry microcarrier for cell culture is 0.43% by mass or more relative to the total amount of the dry microcarrier for cell culture.

Claims

1. A step of immersing alginate microparticle gel containing divalent or higher cations in a gelatin-containing solvent, and The process involves drying the alginate microparticle gel immersed in the aforementioned solvent to form dried microcarriers. A method for producing dry microcarriers for cell culture, which includes a method for culturing cells on a surface, The dry microcarrier for cell culture contains gelatin and alginic acid crosslinked with a divalent or higher cation, and contains gelatin inside the dry microcarrier for cell culture. The gelatin concentration in the solvent into which the alginic acid microparticle gel is immersed is 0.5% by mass or more. Manufacturing method.

2. The manufacturing method according to claim 1, comprising the step of immersing the alginic acid microparticle gel, which has been immersed in the gelatin-containing solvent, in a poor solvent for gelatin before drying.

3. The manufacturing method according to claim 2, wherein the poor solvent is a volatile solvent.

4. The method for producing cells according to claim 1, wherein the dried microcarrier for cell culture comprises a salt of gelatin and alginic acid crosslinked with calcium ions.

5. The manufacturing method according to claim 1, wherein the gelatin content of the entire dry microcarrier for cell culture is 24% by mass or more relative to the total amount of the dry microcarrier for cell culture.

6. The manufacturing method according to claim 1, wherein the nitrogen content in the dry microcarrier for cell culture is 3.9% by mass or more relative to the total amount of the dry microcarrier for cell culture.

7. The manufacturing method according to claim 1, wherein the gelatin contained inside the dried microcarrier for cell culture is bound to alginic acid.

8. A dry microcarrier for cell culture, comprising gelatin and alginic acid crosslinked with divalent or higher cations, for culturing cells on a surface. Let α be the N 1s / C 1s (atomic % / atomic %) of the dried microcarrier for cell culture obtained by X-ray photoelectron spectroscopy (XPS). When the nitrogen content (mass%) of the dried microcarrier for cell culture obtained by CHN elemental analysis or reduced-pressure chemiluminescence is denoted as β, The ratio of β (before grinding) / α (before grinding) is 27.04 or higher, or α(after grinding) / α(before grinding) is 0.43 or higher. (Here, "α (before grinding)" and "β (before grinding)" represent the N 1s / C 1s (atomic % / atomic %) and nitrogen content (mass %) of the dried microcarrier for cell culture, respectively, and "α (after grinding)" represents the N 1s / C 1s (atomic % / atomic %) of the powder obtained by grinding the dried microcarrier for cell culture.) Dry microcarriers for cell culture.

9. The above β (before grinding) / α (before grinding) is 27.04 or more, and The dry microcarrier for cell culture according to claim 8, wherein the ratio of α (after grinding) / α (before grinding) is 0.43 or greater.

10. A dry microcarrier for cell culture, comprising gelatin and alginic acid crosslinked with divalent or higher cations, for culturing cells on a surface. Let α be the N 1s / C 1s (atomic % / atomic %) of the dried microcarrier for cell culture obtained by X-ray photoelectron spectroscopy (XPS). When the nitrogen content (mass%) of the dried microcarrier for cell culture obtained by CHN elemental analysis or reduced-pressure chemiluminescence is denoted as β, The ratio of β (before grinding) to α (before grinding) is 27.04 or higher. α(after grinding) / α(before grinding) is 0.43 or higher. (Here, "α (before grinding)" and "β (before grinding)" represent the N 1s / C 1s (atomic % / atomic %) and nitrogen content (mass %) of the dried microcarrier for cell culture, respectively, and "α (after grinding)" represents the N 1s / C 1s (atomic % / atomic %) of the powder obtained by grinding the dried microcarrier for cell culture.) The average particle size of the aforementioned dried microcarriers for cell culture is 1 mm or less. Dry microcarriers for cell culture.

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