EP-4123011-B1 - AGENT FOR PROMOTING SPHEROID FORMATION

Inventors

• KUBOTA, Munehiro
• TSUJIKAWA, KAZUTAKE

Dates

Publication Date

20260506

Application Date

20210318

Claims (4)

1. A method of producing animal cells in a spheroid form, comprising the steps of: mixing a dispersion liquid of inorganic particles having adsorbability and a cell suspension to obtain a mixture in which the inorganic particles having adsorbability are dispersed, and culturing a cell contained in the obtained mixture to form a spheroid, wherein the solid content concentration of the adsorptive inorganic particles in the dispersion liquid is 0.001 mass% or more and less than 1.000 mass%, wherein the inorganic particles act as spheroid formation promoter, and wherein the inorganic particles are particles composed of at least a swellable layered silicate, wherein the solid content concentration of the swellable layered silicate in the mixture of the cell suspension and the dispersion liquid of the swellable layered silicate is 0.0003 w/v% or more and 0.1667 w/v% or less, wherein an adsorption amount of bovine serum albumin per 1 mg of the inorganic particles having adsorbability is 10 µg or more and 500 µg or less.
2. The method according to claim 1, wherein the inorganic particles are particles composed of at least one kind of swellable layered silicate selected from the group consisting of montmorillonite, hectorite, stevensite, saponite, beidellite, nontrite, sauconite, and swellable mica.
3. The method according to claim 1 or 2, wherein the animal cell is a cell collected from an animal, a cell obtained by culturing a cell collected from an animal, a cell obtained by subjecting a cell collected from an animal to various treatments, or a cultured cell line, and/or wherein the cell is a mammalian animal cell.
4. The method according to any one of claims 1 to 3, wherein the animal cell is cultured in a culture plate for suspension cells to form a spheroid.

Description

TECHNICAL FIELD The present invention relates to a method of producing animal cells in a spheroid form. BACKGROUND ART In recent years, animal cells of mammalians represented by human beings have been used as research materials for a wide variety of studies. In plane culture (two-dimensional cell culture) of animal cells, which has been performed for a long time, cells expand two-dimensionally and proliferate. Thus, a certain culture area is required to obtain a certain number of cultured cells. Further, since the cultured cells are monolayer cells spreading in a two-dimensional direction, there is a problem that the function of the cells themselves cannot necessarily reproduce the state in vivo. Thus, three-dimensionally culturing cells utilizing an original aggregation reaction of adherent animal cells is widely desired. A spheroid (aggregate) obtained by three-dimensional cell culture exhibit a function different from that of cells obtained by two-dimensional cell culture, realize a cell environment closer to a living body, and have high cell activity. Thus, spheroids are useful for drug screening in drug discovery, toxicity tests, construction of a safety evaluation model as an alternative to animal experiments and the like, and are also very important as a biomaterial from which practical information can be obtained. In general three-dimensional cell culture methods, control of the adhesion of cells is important, and scaffold materials, culture vessels, media and the like that are suitably used in three-dimensional cell culture methods have been developed. For example, porous membranes and hydrogels used as scaffold materials are commercially available, and Patent Literature 1 describes a method of culturing cells using a chitin gel produced by gelating chitosan by treating the chitosan with an acetylating agent as a culture base material. Patent Literature 2 describes a base material for cell culture in which a chimeric protein having a hydrophobic material binding site and a cell adhesion site is immobilized on a fiber surface of a fiber structure having a specific average fiber diameter and formed of a polymer compound. Further, Non-Patent Literature 1 describes control of adhesion of cells using a hydrogel in which an inorganic layered compound is present in a polymer matrix. Regarding culture vessels, a plate subjected to microfabrication for the purpose of non-adhesion treatment, a U-bottom plate designed to collect cells and the like can be used for three-dimensional cell culture methods, and are also commercially available. Further, Patent Literature 3 describes that spheroid formation is promoted by culturing a mixture of a cell sample and a polymer obtained by polymerization of one or more selected from the group consisting of D-glucosamine, D-galactosamine, D-glucuronic acid, L-iduronic acid, and D-galactose in a vessel for spheroid formation culture. Patent Literature 4 describes a method of producing a spheroid including the step of culturing cells in the presence of an exogenous cell aggregating agent containing any of an antibody, a lectin, and a cell adhesion molecule. As described above, three-dimensional cell culture can be performed by using special scaffold materials, culture vessels, or culture media. However, such scaffold materials, culture vessels, or culture media are generally expensive. Thus, when a large amount of spheroids is required for, for example, screening for drugs in drug discovery, screening for drug efficacy in primary culture of clinical specimens, and production of useful proteins, the cost burden is large. Further, development of a three-dimensional cell culture method having a high spheroid formation rate and excellent versatility without limitation of target biological tissues or cell types is also desired. A swellable layered silicate such as smectite is a kind of clay minerals, and has a property of swelling and becoming viscous when absorbing a liquid such as water. Smectite is composed of nanosheets spreading in two dimensions. When smectite is swollen with and dispersed in water, a colloidal dispersion liquid having viscosity and thixotropy is obtained. Smectite exists in a state of being separated up to a unit layer in a dispersion liquid. The crystal layer itself of smectite in the dispersion liquid has a permanent negative charge, and cations such as sodium ions are incorporated into the crystal layer to compensate for the permanent negative charge. By utilizing such properties, swellable layered silicates such as smectite have been industrially widely used since a long time ago. For example, a protein crystal formation controlling agent containing a layered silicate compound containing a fluorine atom is described in Patent Literature 5. Further, an agent for searching for protein crystallization conditions, containing a water-swellable layered silicate having a fluorine atom and a hydroxyl group, wherein the fluorine atom is covalently bonded to the