Cell Culture Scaffold Materials

Abstract

To provide a scaffold material suitably used for cell culture.SOLUTION: A scaffold material for cell culture contains bacterial cellulose produced by bacteria and has the following (a) physical properties: (a) a transmittance of light with a wavelength of 500 nm is 35% or more at a solid concentration of 0.1±0.006%(w / v).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a cell culture scaffold material and techniques related thereto. [Background technology]

[0002] Matrigel (Matrigel™ Basement Membrane Matrix, Corning, NY, USA) is widely used as a cell culture scaffold material (Non-Patent Document 1). Matrigel is extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, and contains laminin as its main component, as well as type IV collagen, heparin sulfate proteoglycan, entactin / nidogen, and a number of growth factors. As described above, since Matrigel contains animal-derived components, there is a problem in that its use is restricted.

[0003] It is also known that spheroids (aggregates) can be formed by culturing cells in a medium containing deacylated gellan gum (Patent Document 1). However, this method does not provide sufficient efficiency in forming spheroids. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Graves et al., Breast Cancer Research (2016) 18:11, pp. 1-17 [Patent documents]

[0005] [Patent Document 1] Patent No. 5629893 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a scaffold material that is suitable for use in cell culture. [Means for solving the problem]

[0007] As a result of intensive research to solve the above problems, the present inventors have found that bacterial cellulose produced by bacteria is suitable as a cell culture scaffold material. For example, the present inventors have found that when bacterial cellulose is used as a cell culture scaffold material, spheroids (aggregates) of various cells can be easily formed, and the spheroids can be grown (e.g., increased in number and / or size) with the passage of culture time. The present inventors have also found that when bacterial cellulose is used, the spheroid growth rate is excellent as in the case of using Matrigel, and the spheroid formation efficiency is excellent compared to the case of using gellan gum. Furthermore, the present inventors have found that the activity of various drug metabolizing enzymes (CYPs) is increased in cultures (spheroids) of liver cancer cells in the presence of bacterial cellulose compared to cultures in the absence of bacterial cellulose. Furthermore, the present inventors have found that when stem cells (e.g., iPS cells) are cultured in the presence of bacterial cellulose, they can form spheroids while remaining undifferentiated, and can be induced to differentiate into various cells (e.g., endothelial cells). Based on these findings, the present inventors conducted further studies and completed the present invention.

[0008] The present invention includes the following aspects. [1] A scaffold material for cell culture, comprising bacterial cellulose produced by bacteria, and having the following physical property (a): (a) The transmittance of light with a wavelength of 500 nm is 35% or more at a solids concentration of 0.1±0.006% (w / v). [2] The scaffolding material described in [1], wherein the bacterial cellulose is produced by the bacterium by assimilating one or more of the group consisting of sugar, sucrose-containing by-products produced during sugar production and their hydrolysates, and isomerized sugar. [3] The scaffold material described in [1] or [2], wherein the bacterial cellulose is produced by assimilating one or more selected from the group consisting of glucose, fructose, and molasses. [4] The scaffold material according to any one of [1] to [3], wherein the bacterium is Gluconacetobacter intermedius. [5] The scaffold material according to any one of [1] to [4], wherein the bacterium is Gluconacetobacter intermedius SIID9587 strain (accession number NITE BP-01495). [6] The scaffold material according to any one of [1] to [5], wherein the culture is a static culture. [7] The scaffold material according to any one of [1] to [5], wherein the culture is a suspension culture. [8] The scaffold material according to any one of [1] to [5], wherein the culture is a three-dimensional culture. [9] The scaffold material according to any one of [1] to [8], wherein the cells are cancer cells.

[10] The scaffold material according to any one of [1] to [8], wherein the cells are stem cells.

[11] The scaffold material according to any one of [1] to [8], wherein the cells are primary cultured cells.

[12] A medium composition comprising the scaffold material according to any one of [1] to

[11] .

[13] A coating agent for a cell culture vessel, comprising the scaffold material according to any one of [1] to

[11] .

[14] A method for producing a cell spheroid, comprising the step of culturing cells in the presence of the scaffold material described in any one of [1] to

[11] . Effect of the Invention

[0009] The present invention provides a scaffold material suitable for cell culture. By using the scaffold material of the present invention, for example, spheroids of various cells can be easily formed, and the spheroids can be grown (e.g., increased in number and / or size) with the passage of culture time. In addition, the scaffold material of the present invention is excellent in terms of spheroid growth rate and formation efficiency. Furthermore, when liver cancer cells are cultured in the presence of the scaffold material of the present invention, the activity of various drug metabolizing enzymes (CYP) can be increased. Furthermore, when stem cells (e.g., iPS cells) are cultured in the presence of the scaffold material of the present invention, spheroids can be formed while remaining undifferentiated, and differentiation can be induced into various cells (e.g., endothelial cells). [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows micrographs of the formation of spheroids when various cancer cells were cultured on the scaffold material of the embodiment and when they were cultured without using the scaffold material. [Diagram 2] FIG. 2 is a graph showing the change in size or number of spheroids over time when various cancer cells were cultured on the scaffold material of the example. [Diagram 3] FIG. 3 is a micrograph of MCF-7 human breast cancer cells cultured on the scaffold material of the embodiment and then immunostained. [Figure 4] FIG. 4 is a set of micrographs comparing the growth rates of spheroids of MCF-7 human breast cancer cells cultured on the scaffold material of the embodiment and on Matrigel. [Diagram 5] FIG. 5 is a graph comparing the activity of CYP molecular species when HepG2 human liver cancer cells are cultured on the scaffold material of the embodiment and when they are cultured without using the scaffold material. [Figure 6] FIG. 6 shows micrographs of various cancer cells cultured in suspension in a medium composition containing a scaffold material of the example. [Figure 7]FIG. 7 shows micrographs of the formation of spheroids when induced pluripotent stem cells (iPS cells) were cultured on a scaffold material of the embodiment and when they were cultured without using the scaffold material. [Figure 8] FIG. 8 shows micrographs of the expression of undifferentiated markers when induced pluripotent stem cells (iPS cells) were cultured on the scaffold material of the example and when they were cultured without using the scaffold material. [Figure 9] FIG. 9 shows micrographs of three-dimensional culture using scaffold materials with different dispersant concentrations. [Figure 10] FIG. 10 shows micrographs of suspension culture using scaffold materials with different dispersant concentrations. [Figure 11] FIG. 11 shows micrographs of various cancer cells cultured in suspension in a medium composition containing the scaffold material of the examples or gellan gum. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] [Scaffolding materials for cell culture] A scaffold material for cell culture (hereinafter may be simply referred to as a "scaffold material") contains bacterial cellulose produced by bacteria (hereinafter may be simply referred to as "bacterial cellulose").

[0012] The cells are not particularly limited and any cells can be used, for example, cells derived from animals or plants. The cells are preferably cells derived from animals. Examples of animals include mammals, and specific examples of mammals include humans, rats, mice, guinea pigs, rabbits, dogs, cats, cows, sheep, goats, pigs, and monkeys.

[0013] The cells may be cells derived from any tissue or organ, including, for example, skin, bone, cartilage, vascular tissue, muscle, brain, eye, nerve, lung, thymus, mammary gland, stomach, colon, small intestine, large intestine, heart, pancreas, kidney, spleen, liver, uterus, ovary, testis, bladder, prostate, and oral mucosa.

[0014] In one embodiment, the cell is preferably one selected from germ cell, somatic cell, stem cell and cancer cell.The cell may be a primary cultured cell (e.g., primary hepatocyte; primary stem cell such as primary ES cell, primary iPS cell), a subcultured cell, an established cell line, or a modified (e.g., genetically modified) cell.

[0015] Germ cells include, for example, oogonia, oocytes, spermatogonia, spermatocytes, and the like.

[0016] Examples of somatic cells include fibroblasts, bone marrow cells, immune cells (B lymphocytes, T lymphocytes, neutrophils, macrophages, monocytes, etc.), organ-derived cells (hepatocytes, spleen cells, pancreatic cells, kidney cells, embryonic cells, etc.), muscle tissue cells (skeletal muscle cells, smooth muscle cells, myoblasts, cardiac myocytes, etc.), nerve cells, glial cells, osteoblasts, chondrocytes, endothelial cells, epithelial cells, epidermal cells, interstitial cells, adipocytes, and precursor cells thereof.

[0017] Examples of stem cells include embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, and mesenchymal stem cells.

[0018] Regarding cancer cells, examples of cancer types include brain tumors, tongue cancer, laryngeal cancer, lung cancer, breast cancer, stomach cancer, colorectal cancer (colon cancer, rectal cancer, etc.), esophageal cancer, pancreatic cancer, squamous cell cancer, kidney cancer, liver cancer, bile duct cancer, cervical cancer, endometrial cancer, ovarian cancer, testicular cancer, prostate cancer, bladder cancer, leukemia, and osteosarcoma.

[0019] The method of culturing the cells can be appropriately selected depending on the type of cells, etc. The culture can be static culture or suspension culture. The culture can be culture without using feeder cells and / or cell adhesion proteins. The culture can be two-dimensional culture (plate culture) or three-dimensional culture, but three-dimensional culture is preferable. The scaffold material can be particularly preferably used for the formation of spheroids and further the growth of spheroids (e.g., increase in number and / or size).

[0020] The number of cells to be seeded at the start of the culture is, for example, 1 x 10 3 pieces / cm 2 Or 1×10 3 / mL or more, preferably 1 × 10 4 pieces / cm 2 Or 1×10 4 ≥ 1×10 6 pieces / cm 2 Or 1×10 6 / mL or less, preferably 1 × 10 5 pieces / cm 2 Or 1×10 5 cells / mL or less.

[0021] The culture temperature is not particularly limited, but is, for example, 36 to 38°C, and preferably about 37°C.

[0022] The culture atmosphere is not particularly limited, and may be, for example, a mixed gas of air and carbon dioxide gas. The ratio of carbon dioxide gas in the mixed gas may be, for example, about 5% (v / v).

[0023] The culture period is not particularly limited, but is, for example, 1 to 15 days, and preferably 2 to 10 days.

[0024] The scaffold material means a material or substrate that functions as a scaffold for cells in cell culture. The scaffold material is a material used together with a culture medium, and may be a material used separately from the culture medium (for example, the scaffold material and the culture medium are added to a cell culture vessel in this order without mixing, so that the scaffold material and the culture medium are present as separate layers), or may be a material used by mixing or dispersing in the culture medium.

[0025] The scaffold material usually contains a liquid medium in addition to bacterial cellulose. Examples of the liquid medium include water and organic solvents. Examples of the organic solvent include alcohols (e.g., methanol, ethanol, isopropyl alcohol), ethers (e.g., tetrahydrofuran), ketones (e.g., acetone), amides (e.g., dimethylformamide), and nitriles (e.g., acetonitrile). The liquid medium may be one type alone or two or more types in combination. The form of the scaffold material may be, for example, a liquid, a gel, or the like.

[0026] The solid content of the scaffold material is, for example, 0.1% (w / v) or more, preferably 0.5% (w / v) or more, and, for example, 2.5% (w / v) or less, preferably 2% (w / v) or less. The solid content of the scaffold material can be calculated, for example, by measuring the weight of the scaffold material in an absolute dry state. The bacterial cellulose content of the scaffold material can also be selected from a similar range.

[0027] The scaffold material has a physical property (a) of 35% or more transmittance at 500 nm wavelength at a solid content concentration of 0.1±0.006% (w / v). Here, the transmittance at 500 nm wavelength at a solid content concentration of 0.1±0.006% (w / v) can be, in addition to 35%, for example, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 35% to 99% or less, 36% to 99% or less, 37% to 99% or less, 38% to 99% or less, 40% to 99% or less, 35% to 95% or less, 36% to 95% or less, 37% to 95% or less, 38% to 95% or less, 40% to 99% or less, 40% to 99% or less, 40% to 99% or less, 40% to 99% or less, 40% to 95 ... Examples include 0% or more and 95% or less, 35% or more and 90% or less, 36% or more and 90% or less, 37% or more and 90% or less, 38% or more and 90% or less, 40% or more and 90% or less, 35% or more and 85% or less, 36% or more and 85% or less, 37% or more and 85% or less, 38% or more and 85% or less, 40% or more and 85% or less, 35% or more and 80% or less, 36% or more and 80% or less, 37% or more and 80% or less, 38% or more and 80% or less, and 40% or more and 80% or less.

[0028] The transmittance can be measured, for example, by adjusting the solid concentration of the scaffold material to 0.1±0.006% (w / v), adding 1 mL to a cell, and subjecting it to a spectrophotometer (U-2001 double beam spectrophotometer; Hitachi, Ltd.). Note that a polystyrene disposable cuvette (semi-micro, optical path length 10 mm, optical path width 4 mm) is used as the cell, and ultrapure water can be used as the reference.

[0029] Bacterial cellulose may have a larger average molecular weight than plant-derived cellulose such as cellulose nanofiber derived from pulp. The average molecular weight of cellulose can be measured, for example, using a chromatogram of gel permeation chromatography as an index, and the larger the retention volume of the peak top of the chromatogram, the smaller the average molecular weight, and vice versa. That is, bacterial cellulose may have physical properties such that the retention volume of the peak top of a chromatogram of gel permeation chromatography performed under the following conditions i) to vi) is 2.5 mL or more and less than 3.0 mL. i) the column had an internal diameter of 6.0 mm and a length of 15 cm, packed with a methacrylate polymer with a particle size of 9 μm; ii) the guard column has an inner diameter of 4.6 mm and a length of 3.5 cm; iii) the column temperature is 35°C; iv) The liquid delivery rate is 0.07 mL / min; v) the eluent is a 40-42% (w / w) aqueous solution of tetrabutylphosphonium hydroxide; vi) The final concentration of solids in the eluent is 0.2% (w / v).

[0030] Bacterial cellulose may have a high aspect ratio compared to plant-derived cellulose such as pulp-derived cellulose nanofibers. In one embodiment, the aspect ratio of bacterial cellulose may be 500 or more, 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, 6000 or more, 7000 or more, 8000 or more, 9000 or more, or 10000 or more.

[0031] The fiber width of bacterial cellulose is, for example, 1 to 1000 nm, preferably 2 to 500 nm, more preferably 3 to 100 nm, and further preferably 5 to 70 nm. In one embodiment, the bacterial cellulose may be one in which the proportion of fiber widths of 20 to 30 nm is 80% or more (e.g., 85 to 95%), preferably 90% or more. The fiber width can be measured by observation using an atomic force microscope (AFM).

[0032] Bacterial cellulose can form a fine network structure and has high strength, and also has high water retention and high biocompatibility.

[0033] Bacterial cellulose can be produced, for example, by culturing bacteria in a medium containing a carbon source to produce bacterial cellulose.

[0034] Examples of carbon sources include monosaccharides such as glucose and fructose, disaccharides such as sucrose, maltose, and lactose, oligosaccharides, sugar, sucrose-containing by-products produced during sugar production, their hydrolysates, isomerized sugar, starch hydrolysates, and other sugars, as well as mannitol, ethanol, acetic acid, citric acid, glycerol, and the like, and can be appropriately selected depending on the type of bacteria, culture conditions, production costs, etc.

[0035] Sugar refers to a sweetener whose main component is sucrose (Kojien, 6th edition). It may be chemically synthesized or may be manufactured from natural materials such as sugar cane, sugar beet, sugar maple, sugar palm, and sweet sorghum. Examples of sugar include molasses sugar such as brown sugar, white sugar, cassonade (brown sugar), wasanbon, and maple sugar, and fractionated sugar such as raw sugar and refined sugar. Examples of refined sugar include processed sugar such as white double sugar, medium double sugar, and granulated sugar, car sugar such as white sugar and brown sugar, processed sugar such as cube sugar, rock sugar, powdered sugar, and granulated sugar, as well as liquid sugar.

[0036] Sucrose-containing by-products produced during sugar production refer to by-products produced in the sugar production process that contain sucrose. Specific examples include the pomace of natural raw materials such as sugar cane and sugar beet, as mentioned above, molasses, and residues produced in the refining process using filtration or ion exchange resins.

[0037] Hydrolysates of disaccharides, oligosaccharides, sugar, and sucrose-containing by-products produced during the production of sugar refer to products obtained by subjecting disaccharides, oligosaccharides, sugar, and sucrose-containing by-products produced during the production of sugar to hydrolysis treatment, such as heating in an acidic solution.

[0038] The carbon source is preferably at least one selected from glucose, fructose, and molasses.

[0039] The proportion of the carbon source in the medium is, for example, 1% (w / v) or more, preferably 5% (w / v) or more, and for example, 10% (w / v) or less, preferably 5% (w / v) or less.

[0040] The medium components other than the carbon source preferably contain a dispersant. Examples of the medium include a carbon source, a dispersant, a nitrogen source, inorganic salts, and, if necessary, organic trace nutrients such as amino acids and vitamins.

[0041] Examples of dispersants include hydrophilic dispersants such as CMC (carboxymethyl cellulose), and amphipathic dispersants such as HEC (hydroxyethyl cellulose) and HPC (hydroxypropyl cellulose). Among these, amphipathic dispersants such as HEC and HPC are preferred from the viewpoint of the formation and growth of spheroids.

[0042] The proportion of the dispersant in the culture medium is, for example, 0.1% (w / v) or more, preferably 0.5% (w / v) or more, more preferably 1% (w / v) or more, even more preferably 1.5% (w / v) or more, and for example, 3% (w / v) or less, preferably 2.5% (w / v) or less.

[0043] Examples of the nitrogen source include organic or inorganic nitrogen sources such as ammonium salts, such as ammonium sulfate, ammonium chloride, and ammonium phosphate, nitrates, urea, and peptone.

[0044] The proportion of the nitrogen source in the medium is, for example, 0.1% (w / v) or more, preferably 0.5% (w / v) or more, and for example, 2% (w / v) or less, preferably 1% (w / v) or less.

[0045] Examples of inorganic salts include phosphate salts, magnesium salts, calcium salts, iron salts, and manganese salts.

[0046] The proportion of inorganic salts in the medium is, for example, 0.005% (w / v) or more, preferably 0.01% (w / v) or more, more preferably 0.05% (w / v) or more, even more preferably 0.1% (w / v) or more, particularly preferably 0.2% (w / v) or more, and is, for example, 1% (w / v) or less, preferably 0.5% (w / v) or less.

[0047] Examples of organic micronutrients include amino acids, vitamins, fatty acids, nucleic acids, and further, peptones containing these nutrients, casamino acids, yeast extracts, soy protein hydrolysates, etc. When an auxotrophic mutant strain that requires amino acids for growth is used, the required nutrients can be further supplemented.

[0048] The bacteria are not particularly limited as long as they can produce bacterial cellulose, but are preferably bacteria that can produce bacterial cellulose by stirring culture or aeration culture.Specific examples of the bacteria include Acetobacter bacteria, Gluconacetobacter bacteria, Pseudomonas bacteria, Agrobacterium bacteria, Rhizobium bacteria, and Enterobacter bacteria, and more specifically, Gluconacetobacter intermedius, Gluconacetobacter hansenii, Gluconacetobacter swingsii, Acetobacter pasteurianus, Acetobacter aceti, Acetobacter xylinum, Acetbacter xylinum subsp.sucrofermentans, Acetbacter xylinum subsp.nonacetoxidans, Acetobacter ransens, Sarcina ventriculi, Bacterium xyloides, and Enterobacter sp., and among these, Gluconacetobacter intermedius is preferred. More specifically, examples of the bacterium include Gluconacetobacter intermedius SIID9587 strain (NEDO-01 strain) (accession number NITE BP-01495), Gluconacetobacter xylinus ATCC53582 strain, Gluconacetobacter hansenii ATCC23769 strain, Gluconacetobacter xylinus ATCC700178 (BPR2001) strain, Gluconacetobacter swingsii BPR3001E strain, Acetobacter xylinum JCM10150 strain, and Enterobacter sp. CJF-002 strain. Of these, Gluconacetobacter intermedius SIID9587 strain (NEDO-01 strain) (accession number NITE BP-01495) is preferred.

[0049] Examples of the culture method include stationary culture and agitation culture. Specific examples of the agitation culture include culture without aeration using a fermenter (aeration-free agitation culture), culture with aeration using a fermenter (aeration agitation culture), culture with shaking from side to side using a bladed flask (shaking culture), and rotary culture using a bladed flask (rotation culture). The culture conditions can be the known culture conditions used for culturing the above-mentioned bacteria, and examples of the culture conditions include aeration volume of 1 to 10 L / min, rotation speed of 100 to 800 rpm, temperature of 20 to 40°C, and culture period of 1 to 7 days.

[0050] In the production of bacterial cellulose, if necessary, a carbon source pretreatment step, a pre-preculture step, a preculture step, purification of bacterial cellulose, drying, suspension step, etc. can be performed. Bacterial cellulose can be produced, for example, according to the description in Japanese Patent No. 5752332, Cellulose (2013) 20:2971-2979, etc.

[0051] The scaffold material usually contains a dispersant used in the production of bacterial cellulose, and the solid content of the scaffold material usually consists of bacterial cellulose and a dispersant. The ratio of the dispersant to the solid content of the scaffold material is, for example, 1% (w / w) or more, preferably 3% (w / w) or more, and, for example, 40% (w / w) or less, preferably 35% (w / w) or less, and more preferably 30% (w / w) or less. By adjusting to such a ratio, it is possible to have moderate hydrophobicity and / or hydrophilicity and to increase the efficiency of spheroid formation. The ratio of the dispersant to the solid content of the scaffold material can be measured by a conventional method. For example, when the dispersant is CMC, it can be measured according to the quantitative method of carmellose sodium of the Pharmaceutical Excipients Standard, and when the dispersant is HPC, it can be measured according to the quantitative method of hydroxypropyl cellulose of the Japanese Pharmacopoeia.

[0052] [Medium composition] The medium composition is not particularly limited as long as it contains the bacterial cellulose or the scaffold material. The medium of the medium composition usually includes basic components containing a carbon source, a nitrogen source, inorganic salts, and vitamins. Examples of such basic components include Minimum Essential Medium (MEM), Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium (DMEM), Eagle's Minimal Essential Medium (EMEM), Minimum Essential Medium alpha Modification (α-MEM), Iscove's Modified Dulbecco's Medium (IMDM), Glasgow Minimum Essential Medium (GMEM), Ham's F12 Medium (F12), DMEM / F12, RPMI1640, L-15, McCoy's 5A, 199, Richter CM, ReproCell products (e.g., Primate ES Cell Medium, ReproStem, ReproFF, ReproFF2, ReproXF), and PromoCell products (e.g., hPSC Growth medium DXF). The basic component may contain other components (eg, serum such as fetal bovine serum (FBS); growth factors such as FGF; antibiotics such as penicillin and streptomycin; Y-27632).

[0053] The proportion of the scaffold material (solid content) in the medium composition is, for example, 0.05% (w / v) or more, preferably 0.1% (w / v) or more, and, for example, 1% (w / v) or less, preferably 0.5% (w / v) or less. The proportion of bacterial cellulose in the medium composition can also be selected from a similar range.

[0054] The medium composition may be sterilized. The medium composition is resistant to high temperatures and will not decompose even if subjected to autoclave sterilization or dry heat sterilization, and therefore may be sterilized by autoclave or dry heat.

[0055] [Cell culture vessel coating agent] The coating agent for the cell culture vessel is not particularly limited as long as it contains the bacterial cellulose or the scaffold material. The coating agent is usually applied to the cell culture surface of the cell culture vessel and used to form a scaffold for cell culture.

[0056] Examples of the shape of the cell culture vessel include a dish, a multi-well plate, a flask, a cell insert, and a membrane.

[0057] Examples of materials for the cell culture vessel include polymers, glass, modified glass, ceramics, metals, etc. Examples of polymers include polyethylene terephthalate, polyethylene naphthalate, polystyrene, polycarbonate, triacetyl cellulose, polyimide, nylon, polyethylene, polypropylene, vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyethersulfone, polylactic acid, polyglycolic acid, polycaprolactone, etc.

[0058] The proportion of the scaffold material (solid content) in the coating agent is, for example, 0.1% (w / v) or more, preferably 0.5% (w / v) or more, and, for example, 2.5% (w / v) or less, preferably 2% (w / v) or less. The proportion of bacterial cellulose in the coating agent can also be selected from the same range.

[0059] The coating agent may be sterilized. The coating agent is resistant to high temperatures and will not decompose even if subjected to autoclave sterilization or dry heat sterilization, and therefore may be sterilized by autoclave or dry heat.

[0060] [How to manufacture cell spheroids] The method for producing cell spheroids comprises culturing cells in the presence of the bacterial cellulose or the scaffold material. The cells and culture conditions can be selected from those described for the scaffold material.

[0061] The diameter (equivalent circle diameter) of the spheroids is not particularly limited, but is, for example, 40 μm or more, preferably 50 μm or more, and is, for example, 1,500 μm or less, preferably 1,000 μm or less. The number of spheroids is not particularly limited, but is, for example, 100 pieces / cm. 2 Or 100 cells / mL or more, preferably 200 cells / cm 2 Or 200 cells / mL or more, for example 2,000 cells / cm 2 Or 5,000 cells / mL or less, preferably 1,500 cells / cm 2 Or less than 3,000 cells / mL. EXAMPLES

[0062] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0063] Preparation of cell suspension Colon26 mouse colon cancer cells (RIKEN BRC, RCB2657), HepG2 human liver cancer cells (RIKEN BRC, RCB1648), MCF-7 human breast cancer cells (RIKEN BRC, RCB1904), 4T1 mouse breast cancer cells (JCRB cell bank, JCRB1447), and MKN45 human gastric cancer cells (RIKEN BRC, RCB1001) were seeded on the dishes and cultured in a 5% CO2, 37°C incubator. After that, 1.0 × 10 5 RPMI medium (containing 10% FBS and penicillin / streptomycin) was added to prepare a cell suspension at 1000 cells / mL. The number of cells in the cell suspension was counted using a Countess II Automated Cell Counter (Thermo Fisher Scientific, MA, USA).

[0064] Preparation of scaffold material For pre-culture and main culture, HS (Hestrin-Schramm) medium (composition: bacto peptone 0.5 w / v%, yeast extract 0.5 w / v%, Na2HPO4 0.27 w / v%, citric acid 0.115 w / v%, carbon source 2 w / v%) supplemented with 2 w / v% CMC was prepared and used. First, the NEDO-01 strain (G. intermedium SIID9587 strain) was precultured using HS medium (carbon source: glucose) to grow the bacteria. Next, the preculture solution was inoculated into 5 L of HS medium (carbon source: molasses), and the main culture was performed by aeration and agitation culture for 50-72 hours using a fermenter (manufactured by Takasugi Seisakusho) under conditions of aeration volume of 1 vvm, rotation speed of 100-250 rpm, and temperature of 30°C. A 1.0 w / v% NaOH aqueous solution was added to the culture solution obtained by the main culture (main culture solution) and agitated at 70°C and 100 rpm for 2 hours to remove the bacterial components. The lysis was observed with AFM to confirm the presence or absence of remaining bacteria. Pure water was added thereto and centrifuged, and the supernatant was removed. This operation was repeated until the pH of the precipitate became 7 or less in a wet state, and the product was purified to produce NFBC-CMC with a solid concentration of 1.0 w / v%. The concentration was adjusted by measuring the weight of NFBC-CMC (sol) after heating at 105°C for 2 hours (absolutely dry state) and diluting the NFBC-CMC (sol) with water. 1.0 w / v% NFBC-CMC was sterilized by high-pressure steam (121°C, 20 minutes). 0.5 w / v% NFBC-CMC was prepared by diluting the sterilized NFBC-CMC with ultrapure water (CMC concentration in solids: 13.6 ± 0.3 w / w%). In addition, 0.5 w / v% NFBC-HPC was prepared in the same manner except that CMC was replaced with HPC (HPC concentration in solids: 12.7 ± 2.9 w / w%). The transmittance of light with a wavelength of 500 nm was measured for these NFBC-CMC and NFBC-HPC by the following method.

[0065] (Transmittance measurement) The final solid concentration of NFBC-CMC or NFBC-HPC was adjusted to 0.1±0.006 w / v%, and then 1 mL of each was added to a cell and subjected to a spectrophotometer (U-2001 double beam spectrophotometer; Hitachi, Ltd.) to measure the transmittance of light with a wavelength of 500 nm. A disposable polystyrene cuvette (semi-micro, optical path length 10 mm, optical path width 4 mm) was used as the cell, and ultrapure water was used as the reference. The transmittance was calculated by averaging the above measurement repeated three times.

[0066] (Transmittance results) The transmittance of NFBC-CMC was 72.3±4.0%, and that of NFBC-HPC was 70.9±1.8%.

[0067] 3D culture of cells using NFBC-HPC 500 μL or 250 μL of 0.5 w / v% NFBC-HPC was added to a 12-well plate or a 24-well plate, respectively, and allowed to stand. 5 1 mL or 500 μL of each of the NFBC-HPC cells (cells / mL) was slowly dropped onto the NFBC-HPC. After that, the cells were cultured at 37°C and 5% CO2 for several days, and the spheroids were observed and photographed using a HS all-in-one fluorescence microscope BZ-9000 (KEYENCE, Osaka, Japan) or a routine inverted microscope Primo Vert (ZEISS, Oberkochen, Germany).

[0068] result All cancer types (Colon26, HepG2, MCF-7, 4T1, and MKMN45) formed spheroids when cultured on NFBC-HPC (3D culture) (Figure 1). When cultured in cell suspension alone without adding NFBC-HPC (plate culture), all cells showed only lateral growth and did not form spheroids.

[0069] Quantification of spheroid size and number The size and number of spheroids were quantified using image processing software (ImageJ). Cell clusters with a size of 40 μm or more were evaluated as spheroids.

[0070] result The growth of the spheroids was evaluated based on the changes in size (diameter) and number of spheroids (Figure 2). HepG2 and Colon26 spheroids showed a tendency for the spheroid diameter to increase depending on the number of days of culture. The number of spheroids (spheroids with a diameter of 40 μm or more) tended to increase depending on the number of days of culture for all spheroids of HepG2, Colon26, 4T1, and MCF-7.

[0071] Immunostaining of spheroids 250 μL of 0.5 w / v% NFBC-CMC was added to each well of a 24-well plate and allowed to stand. 5500 μL of α-Tubulin Monoclonal Antibody (0.5 μg / mL) was slowly dropped onto the NFBC-CMC and cultured at 37°C for several days in a 5% CO2 environment. 1 mL of phosphate buffer solution (PBS (-)) was added to each well and slowly pipetted, after which the entire volume was transferred to a 15 mL tube (Thermo Fisher Scientific) and centrifuged (1,000 rpm, 4°C, 5 to 15 minutes). The cells were washed with PBS (-), suspended in 1 mL of 4% paraformaldehyde, and left to stand at room temperature for 1 hour to fix the cells. The cells were washed with PBS (-), resuspended in 1 mL of 0.5 μg / mL α-Tubulin Monoclonal Antibody (Thermo Fisher Scientific), and then placed on a ROTARY MIXER NRC-20D (NISSIN, Tokyo, Japan) and reacted at room temperature for 24 hours while rotating. The cells were washed with PBS (-), resuspended in 1 mL of 10 μg / mL Hoechst 33342 (DOJINDO, Kumamoto, Japan), and allowed to stand at room temperature for 2 hours to react. The cells were washed with PBS (-), and resuspended in 1 mL of PBS (-). 160 μL of the prepared spheroid suspension was dropped onto an IWAKI GLASS BASE DISH (AGC Techno Glass Co., Ltd., Shizuoka, Japan), covered with a cover glass (Matsunami Glass Industry Co., Ltd., Osaka, Japan), and observed and photographed using a confocal laser microscope LSM700 (ZEISS).

[0072] result The blue color indicates the cell nucleus, and the green color indicates the cytoskeleton α-Tubulin (Figure 3). The spheroids produced had multiple nuclei, shown in blue, indicating that they were composed of a number of cells. In addition, the spheroids produced expressed cytoskeleton proteins such as α-Tubulin, just like normal cells.

[0073] Comparison with 3D culture using Matrigel 250 μL of 0.5 w / v% NFBC-CMC was added to each well of a 24-well plate and allowed to stand. 5500 μL of MCF-7 cell suspension (1.0 × 10 cells / mL) was slowly dropped onto the NFBC-CMC and cultured at 37°C for several days in a 5% CO2 environment. Matrigel (Matrigel™ Basement Membrane Matrix, Corning, NY, USA) was coated onto a 24-well plate according to the recommended protocol. 5 500 μL of each of the 100 cells / mL culture medium was added dropwise and cultured at 37°C in a 5% CO2 environment for several days.

[0074] result When MCF-7 cells were cultured on NFBC-CMC, the number of spheroids increased significantly depending on the number of days of culture (Figure 4). Furthermore, this proliferation rate was almost the same as when Matrigel, which is commonly used for 3D culture, was used.

[0075] Three-dimensional culture of HepG2 cells and evaluation of CYP species activity 250 μL of 0.5 w / v% NFBC-HPC was added to each well of a 24-well plate and allowed to stand. HepG2 cell suspension (1.0 × 10 5500 μL of each of the NFBC-HPC cells (100 cells / mL) was slowly dropped onto the NFBC-HPC and cultured at 37°C for 7 days in a 5% CO2 environment. The culture supernatant was removed, and 500 μL of PBS (-) was added to wash the cells. After removing the supernatant, the activity of each CYP molecular species (CYP3A4, CYP2C9, and CYP2C19) was measured. CYP activity was evaluated using the P450-Glo-CYP3A4-Assay-and-Screening-System (Promega, Tokyo, Japan), P450-Glo-CYP2C9-Assay-and-Screening-System (Promega), or P450-Glo-CYP2C19-Assay-and-Screening-System (Promega) according to the recommended protocol as follows. The substrate reagents for CYP3A4, CYP2C9, and CYP2C19 were diluted 1,000-fold, 50-fold, and 1,000-fold with DMEM, respectively, and 300 μL was added to each well in which the cells were cultured. At this time, an equal amount of each diluted substrate was also added to an empty well as background. The reaction was then carried out for 1 hour, 4 hours, and 2 hours, respectively, at 37°C and 5% CO2. 25 μL of culture supernatant and 25 μL of reaction reagent were added to a 96-well white plate (Greiner Bio One, Cremsmunster, Austria) and left to stand at room temperature for 20 minutes. CYP activity was then evaluated by measuring luminescence intensity using EnSpire (PerkinElmer, MA, USA).

[0076] result CYP activity was evaluated using the HepG2 spheroids (Figure 5). No activity was observed in the CYP molecular species (CYP3A4, CYP2C9, and CYP2C19) in the cells cultured on a plate. On the other hand, in the HepG2 spheroids prepared using NFBC-HPC, the activity of all CYP molecular species was significantly increased.

[0077] Spheroid formation by suspension culture NFBC-HPC was mixed with RPMI medium (containing 10% FBS and penicillin / streptomycin) to a final solid concentration of 0.1 w / v% to prepare NFBC-HPC-containing medium. HepG2 or Colon26 cell suspension was prepared and 1.0 × 10 4 Add NFBC-HPC-containing medium at a concentration of 1000 cells / mL and mix with Nunc TM EasYFlask TM 25cm 2 (Thermo Fisher Scientific) for several days at 37°C in a 5% CO2 environment. During the culture, observations and photographs were performed using a routine inverted microscope Primo Vert (ZEISS).

[0078] result When HepG2 cells or Colon26 cells were cultured in suspension in a medium containing 0.1 w / v% NFBC-HPC, spheroids with a diameter of about 200 to 300 μm could be produced with either cell (Figure 6). Compared to culturing on NFBC-HPC gel, it was possible to produce a large number of spheroids at once by culturing the cells in suspension in a medium containing NFBC-HPC.

[0079] 3D culture of iPS cells Feeder cells SL10 (REPROCELL, Kanagawa, Japan) and iPS cells 201B7 (RIKEN BRC, HPS0063) were stored in liquid nitrogen. SL10 were seeded on a gelatin-coated 60 mm dish and cultured overnight at 37°C and 5% CO2. iPS cells were removed from the liquid nitrogen, quickly seeded on an SL10 culture dish, and then cultured at 37°C and 5% CO2 to plate culture the iPS cells. The differentiated iPS cells were removed at the time of passage, and 1 mL of iPS medium (a mixture of Repro Stem (REPROCELL, Kanagawa, Japan), 10 μg / mL bFGF (REPROCELL), and 10 mM Y-27632 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) in a ratio of 2,000:1:2 (volume ratio)) was added to the collected iPS cell colonies, and the iPS cell suspension was prepared by pipetting so that the colony size was about 100 to 200 μm. 250 μL of 0.9 w / v% NFBC-HPC was added to each well of a 24-well plate and left to stand, and 480 μL of iPS medium was slowly added, followed by 20 μL of the iPS cell suspension, and the plate was cultured at 37°C for several days in a 5% CO2 environment. During the culture, observation and photography were performed using a routine inverted microscope Primo Vert (ZEISS).

[0080] result Compared to plate culture (on-feeder culture) of iPS cells, we demonstrated that culturing iPS cells on NFBC-HPCs formed three-dimensional iPS spheroids (Figure 7). Furthermore, the iPS spheroids produced grew in a culture-day-dependent manner.

[0081] Evaluation of undifferentiated state of iPS spheroids iPS spheroids cultured on NFBC-HPC were collected, washed with PBS (-), and then 1 mL of 4% paraformaldehyde was added and allowed to stand at room temperature for 15 minutes to fix the cells. After washing the cells with PBS (-), 1 mL of 0.1% Triton X-100 solution was added and rotated at room temperature for 15 minutes (ROTARY MIXER NRC-20D), and 1 mL of 3% BSA solution was added and rotated at room temperature for 3 hours. The cells were washed with PBS (-), and 300 μL of Anti Oct3 / 4 antibody (FUJIFILM Wako Pure Chemical Corporation) diluted 500-fold with PBS (-) was added to suspend the cells, and the cells were rotated at room temperature for 8 hours. The cells were washed three times with PBS (-), and then the secondary antibody Alexa Fluor 1000-fold diluted 500-fold with PBS (-) was added to suspend the cells, and the cells were rotated at room temperature for 8 hours. TM The cells were suspended in 300 μL of 488 goat anti-rabbit IgG (H+L) (Thermo Fisher Scientific) and rotated at room temperature for 1 hour in the dark. The cells were washed with PBS (-), and 200 μL of 1,000-fold diluted Hoechst 33342 (DOJINDO) was added to suspend the cells and rotated at room temperature for 10 minutes. The cells were washed with PBS (-), and 300 μL of PBS (-) was added to suspend the cells to prepare an iPS spheroid suspension. 160 μL of the iPS spheroid suspension was added to an IWAKI GLASS BASE DISH (AGC Techno Glass Co., Ltd.), covered with a cover glass (Matsunami Glass Industry Co., Ltd.), and observed and photographed using a confocal laser microscope LSM700 (ZEISS).

[0082] result To evaluate the undifferentiated state of iPS spheroids created using NFBC-HPC, the expression of undifferentiated markers (Oct3 / 4) was observed by immunostaining (Figure 8). Expression of Oct3 / 4 was observed in iPS cells cultured on a plate that maintained the undifferentiated state, and Oct3 / 4 expression was also observed in iPS spheroids created using NFBC-HPC.

[0083] 3D cell culture using NFBC-HPC with different dispersant concentrations The dispersant concentration in the medium during preparation of NFBC-HPC was set to 1.0% or 2.0%, and NFBC-HPC was prepared under each condition. 250 μL of each NFBC-HPC with a solid content concentration of 0.5 w / v% was added to a 24-well plate and allowed to stand. Colon 26 cell suspension (1.0 × 10 5 500 μL of each of the NFBC-HPC cells (cells / mL) was slowly dropped onto the NFBC-HPC. After that, the cells were cultured at 37°C in a 5% CO2 environment for 4 days, and the spheroids were observed and photographed using a routine inverted microscope Primo Vert (ZEISS).

[0084] result When cultured on NFBC-HPC with a dispersant concentration of 1.0%, some spheroids were formed, but the rest fell to the bottom of the well and were cultured as a flat surface (Figure 9). On the other hand, when cultured on NFBC-HPC with a dispersant concentration of 2.0%, the cells were retained on the NFBC-HPC for a long time, and more spheroids were formed.

[0085] Suspension culture of cells using NFBC-HPC with different dispersant concentrations The dispersant concentration in the medium during preparation of NFBC-HPC was set to 1.0% or 2.0%, and NFBC-HPC was prepared under each condition. Each NFBC-HPC was mixed with the medium so that the final solid concentration was 0.1 w / v%, and NFBC-HPC-containing medium was prepared. A cell suspension of Colon 26 was prepared, and 1.0 × 10 4 Add NFBC-HPC-containing medium at a concentration of 1000 cells / mL and mix with Nunc TM EasYFlask TM 25cm 2 (Thermo Fisher Scientific), and cultured for 4 days at 37°C in a 5% CO2 environment, and then observed and photographed using a routine inverted microscope Primo Vert (ZEISS).

[0086] result When cultured in a medium containing NFBC-HPC with a dispersant concentration of 1.0%, some spheroids were formed, but the rest were observed to have fallen to the bottom of the flask (Figure 10).On the other hand, when cultured in a medium containing NFBC-HPC with a dispersant concentration of 2.0%, most of the spheroids formed were observed to be cultured floating in the medium.

[0087] Comparison with suspension culture using gellan gum NFBC-HPC was mixed with the medium so that the final solid concentration of NFBC-HPC was 0.1 w / v%, to prepare a medium containing NFBC-HPC. In addition, gellan gum-containing medium was prepared using FCeM(R) Preparation Kit 500 (Nissan Chemical Industries, Japan). MCF-7 or HepG2 cell suspensions were prepared and 1.0 × 10 4 Add NFBC-HPC-containing medium or gellan gum-containing medium at a concentration of 1000 cells / mL and mix with Nunc TM EasYFlask TM 25cm 2 (Thermo Fisher Scientific), and cultured for 3 to 9 days at 37°C and 5% CO2, and then observed and photographed using a routine inverted microscope Primo Vert (ZEISS).

[0088] result In suspension culture of MCF-7 cells, spheroids with diameters of 200 μm or more were formed in the NFBC-HPC-containing medium, whereas no spheroids were formed in the gellan gum-containing medium (Figure 11). In suspension culture of HepG2 cells, many spheroids with diameters of around 100 μm were observed in the NFBC-HPC-containing medium, whereas the number of spheroids formed in the gellan gum-containing medium was significantly smaller.

Claims

1. A scaffold material for cell culture, comprising a liquid medium which is water, and a solid component consisting of bacterial cellulose produced by bacteria and a dispersant, the bacteria being Gluconacetobacter intermedius strain SIID9587 (accession number NITE P-01495), the scaffold material having the following physical property (a): (a) The transmittance of light with a wavelength of 500 nm is 35% or more at a solids concentration of 0.1±0.006% (w / v).

2. The scaffolding material of claim 1, wherein the bacterial cellulose is produced by the bacterium by assimilating one or more selected from the group consisting of sugar, sucrose-containing by-products produced during sugar production and their hydrolysates, and isomerized sugar.

3. The scaffold material according to claim 1 or 2, wherein the bacterial cellulose is produced by assimilating one or more selected from the group consisting of glucose, fructose, and molasses.

4. The scaffold material according to any one of claims 1 to 3, wherein the culture is a static culture.

5. The scaffold material according to any one of claims 1 to 3, wherein the culture is a suspension culture.

6. The scaffold material according to any one of claims 1 to 3, wherein the culture is a three-dimensional culture.

7. The scaffold material according to any one of claims 1 to 6, wherein the cells are cancer cells.

8. The scaffold material according to any one of claims 1 to 6, wherein the cells are stem cells.

9. The scaffold material according to any one of claims 1 to 6, wherein the cells are primary cultured cells.

10. A medium composition comprising the scaffold material according to any one of claims 1 to 9.

11. A coating agent for a cell culture vessel, comprising the scaffold material according to any one of claims 1 to 9.

12. A method for producing cellular spheroids, comprising culturing cells in the presence of a scaffold material according to any one of claims 1 to 9.

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