JP7599637B2 - Cell Culture Scaffold Materials - Google Patents

Description

NITE P-1495

The present invention relates to a cell culture scaffold material and related technologies.

Matrigel (Matrigel(TM) 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 such, Matrigel contains animal-derived components, which limits its use.

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.

Graves et al., Breast Cancer Research (2016) 18:11, 1-17

Patent No. 5629893

One objective of the present invention is to provide a scaffold material suitable for cell culture.

The present inventors have conducted intensive research to solve the above problems and 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) over the course of culture time. The present inventors have also found that when bacterial cellulose is used, the spheroids have a superior growth rate, similar to when Matrigel is used, and that the spheroid formation efficiency is superior to when gellan gum is used. Furthermore, the present inventors have found that, for liver cancer cells, the culture (spheroid) in the presence of bacterial cellulose has increased activity of various drug metabolizing enzymes (CYPs) compared to when cultured in the absence of bacterial cellulose. Furthermore, the 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 inventors have conducted further studies and completed the present invention.

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 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 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 a step of culturing cells in the presence of the scaffold material according to any one of [1] to [11].

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) over the course 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).

FIG. 1 shows micrographs of the formation of spheroids when various cancer cells were cultured on a scaffold material of the embodiment and when they were cultured without using the scaffold material. 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. FIG. 3 is a micrograph of MCF-7 human breast cancer cells cultured on the scaffold material of the example and then immunostained. FIG. 4 is a set of micrographs comparing the growth rates of spheroids when MCF-7 human breast cancer cells are cultured on the scaffold material of the embodiment and when they are cultured on Matrigel. 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. FIG. 6 shows micrographs of various cancer cells cultured in suspension in a medium composition containing a scaffold material of the example. 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. FIG. 8 shows micrographs of the expression of undifferentiated markers 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. FIG. 9 shows micrographs of three-dimensional culture using scaffold materials with different dispersant concentrations. FIG. 10 shows micrographs of suspension culture using scaffold materials with different dispersant concentrations. 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.

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

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.

The cells may be cells derived from any tissue or organ. Examples of tissues or organs include 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.

In one embodiment, the cells are preferably one selected from germ cells, somatic cells, stem cells, and cancer cells. The cells may be primary cultured cells (e.g., primary hepatocytes; primary stem cells such as primary ES cells and primary iPS cells), subcultured cells, established cell lines, or modified (e.g., genetically modified) cells.

Examples of germ cells include oogonia, oocytes, spermatogonia, and spermatocytes.

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.

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.

In terms of 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.

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 suitable for the formation of spheroids and further the growth of spheroids (e.g., an increase in number and/or size).

The number of cells seeded at the start of culture is, for example, 1 x ¹⁰³ cells/ ^cm2 or 1 x ¹⁰³ cells/mL or more, preferably 1 x ¹⁰⁴ cells/ ^cm2 or 1 x ¹⁰⁴ cells/mL or more, and for example, 1 x ¹⁰⁶ cells/ ^cm2 or 1 x ¹⁰⁶ cells/mL or less, preferably 1 x ¹⁰⁵ cells/ ^cm2 or 1 x ¹⁰⁵ cells/mL or less.

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

The culture atmosphere is not particularly limited, but may be, for example, a mixture of air and carbon dioxide. The proportion of carbon dioxide in the mixture may be, for example, about 5% (v/v).

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

A scaffold material means a material or substrate that functions as a scaffold for cells in cell culture. A scaffold material is a material that is used together with a culture medium, and may be a material that is used separately from the culture medium (for example, by adding the scaffold material and culture medium to a cell culture vessel in that order without mixing, so that the scaffold material and culture medium exist as separate layers), or may be a material that is used by mixing or dispersing in the culture medium.

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.

The percentage of solids in 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 percentage of solids in the scaffold material can be calculated, for example, by measuring the weight of the scaffold material in an absolute dry state. The percentage of bacterial cellulose in the scaffold material can also be selected from a similar range.

The scaffolding material has a physical property (a) of 35% or more transmittance for light with a wavelength of 500 nm at a solid content concentration of 0.1±0.006% (w/v). Here, the transmittance for light with a wavelength of 500 nm at a solid content concentration of 0.1±0.006% (w/v) is, in addition to 35% or more, 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 99% or less, 40% to 99% or less, 40% to 99% or less, 40% to 95 ... % 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, 40% or more and 80% or less, etc.

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 disposable polystyrene 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.

Bacterial cellulose may have a larger average molecular weight than plant-derived cellulose such as pulp-derived cellulose nanofiber. 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 inner 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).

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.

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 even more preferably 5 to 70 nm. In one embodiment, the bacterial cellulose may be bacterial cellulose 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).

Bacterial cellulose, for example, can form a fine network structure and has high strength. In addition, bacterial cellulose can have high water retention and high biocompatibility.

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

Carbon sources include, for example, 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, etc., and can be appropriately selected depending on the type of bacteria, culture conditions, production costs, etc.

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

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

Hydrolyzates of disaccharides, oligosaccharides, sugar, and sucrose-containing by-products produced during sugar production are products obtained by subjecting disaccharides, oligosaccharides, sugar, and sucrose-containing by-products produced during sugar production to hydrolysis, such as heating in an acidic solution.

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

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.

The medium components other than the carbon source preferably contain a dispersant. Examples of the medium include media containing a carbon source, a dispersant, a nitrogen source, inorganic salts, and, as necessary, organic micronutrients such as amino acids and vitamins.

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

The proportion of the dispersant in the 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 is, for example, 3% (w/v) or less, preferably 2.5% (w/v) or less.

Nitrogen sources include organic or inorganic nitrogen sources such as ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, nitrates, urea, and peptone.

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.

Inorganic salts include phosphate salts, magnesium salts, calcium salts, iron salts, manganese salts, etc.

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.

Organic micronutrients include amino acids, vitamins, fatty acids, nucleic acids, and even peptones, casamino acids, yeast extract, soy protein hydrolysates, and the like that contain these nutrients. When using auxotrophic mutants that require amino acids for growth, the required nutrients can be further supplemented.

The bacteria are not particularly limited as long as they are capable of producing bacterial cellulose, but are preferably bacteria capable of producing bacterial cellulose by stirring or aeration culture. Specific examples include Acetobacter bacteria, Gluconacetobacter bacteria, Pseudomonas bacteria, Agrobacterium bacteria, Rhizobium bacteria, and Enterobacter bacteria. More specific examples include 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., among which Gluconacetobacter intermedius is preferred. More specifically, examples of the strain include Gluconacetobacter intermedius SIID9587 (NEDO-01) (accession number NITE BP-01495), Gluconacetobacter xylinus ATCC53582, Gluconacetobacter hansenii ATCC23769, Gluconacetobacter xylinus ATCC700178 (BPR2001), Gluconacetobacter swingsii BPR3001E, Acetobacter xylinum JCM10150, and Enterobacter sp. CJF-002. Of these, Gluconacetobacter intermedius SIID9587 (NEDO-01) (accession number NITE BP-01495) is preferred.

Examples of the culture method include static culture and agitation culture. Specific examples of 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 well-known culture conditions used for culturing the above-mentioned bacteria, such as an aeration rate of 1 to 10 L/min, a rotation speed of 100 to 800 rpm, a temperature of 20 to 40°C, and a culture period of 1 to 7 days.

When producing bacterial cellulose, a carbon source pretreatment step, a pre-preculture step, a preculture step, purification of bacterial cellulose, drying, suspension steps, etc. can be performed as necessary. Bacterial cellulose can be produced, for example, according to the descriptions in Patent No. 5752332 and Cellulose (2013) 20:2971-2979.

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 the ratio in this way, it is possible to obtain a suitable 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 for carmellose sodium in the Pharmaceutical Excipients Standards, and when the dispersant is HPC, it can be measured according to the quantitative method for hydroxypropyl cellulose in the Japanese Pharmacopoeia.

[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).

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.

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, so it may be sterilized by autoclave or dry heat.

[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.

Examples of cell culture vessel shapes include dishes, multi-well plates, flasks, cell inserts, membranes, etc.

Examples of materials for cell culture vessels include polymers, glass, modified glass, ceramics, and metals. 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, and polycaprolactone.

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 a similar range.

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, so it may be sterilized by autoclave or dry heat.

[Method of manufacturing 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.

The diameter (circle equivalent diameter) of the spheroids is not particularly limited, but is, for example, 40 μm or more, preferably 50 μm or more, and 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/ ^cm2 or 100 pieces/mL or more, preferably 200 pieces/ ^cm2 or 200 pieces/mL or more, and for example, 2,000 pieces/ ^cm2 or 5,000 pieces/mL or less, preferably 1,500 pieces/ ^cm2 or 3,000 pieces/mL or less.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

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 dishes and cultured in an incubator with 5% _CO2 at 37°C. Then, RPMI medium (containing 10% FBS and penicillin/streptomycin) was added to prepare a cell suspension at 1.0× ¹⁰⁵ cells/mL. The number of cells in the cell suspension was counted using a Countess II Automated Cell Counter (Thermo Fisher Scientific, MA, USA).

Preparation of scaffold material For pre-culture and main culture, HS (Hestrin-Schramm) medium (composition: bacto pepton 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 was inoculated into 5 L of HS medium (carbon source: molasses), and the main culture was performed using a fermenter (manufactured by Takasugi Seisakusho) under the conditions of aeration volume of 1 vvm, rotation speed of 100-250 rpm, and temperature of 30°C for 50-72 hours. A 1.0 w/v% NaOH aqueous solution was added to the culture solution obtained by the main culture (main culture solution) and stirred 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 in a wet state became 7 or less, 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%). 0.5 w/v% NFBC-HPC was also 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 for these NFBC-CMC and NFBC-HPC was measured by the following method.

(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 used in 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.

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

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 left to stand. 1 mL or 500 μL of cell suspension (1.0 × 10 ⁵ cells/mL) was slowly dropped onto the NFBC-HPC. After that, the plates were cultured at 37°C and 5% CO ₂ 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).

Results: 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 the addition of NFBC-HPC (plate culture), all cells showed only lateral growth and did not form spheroids.

Quantification of size and number of spheroids 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.

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.

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. 500 μL of MCF-7 cell suspension (1.0 × 10 ⁵ cells/mL) was slowly dropped onto the NFBC-CMC and cultured at 37°C and 5% CO ₂ for several days. 1 mL of phosphate buffer solution (PBS (-)) was added to each well and gently 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 allowed 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 incubated at room temperature for 24 hours while rotating on a ROTARY MIXER NRC-20D (NISSIN, Tokyo, Japan). The cells were washed with PBS (-), resuspended in 1 mL of 10 μg/mL Hoechst 33342 (DOJINDO, Kumamoto, Japan), and incubated at room temperature for 2 hours. 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 Co., Ltd., Osaka, Japan), and observed and photographed using a confocal laser microscope LSM700 (ZEISS).

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 several cells. In addition , the spheroids produced expressed cytoskeleton proteins such as α-Tubulin, just like normal cells.

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. 500 μL of MCF-7 cell suspension (1.0×10 ⁵ cells/mL) was slowly dropped onto the NFBC-CMC and cultured at 37°C and 5% CO ₂ for several days. Matrigel (Matrigel™ Basement Membrane Matrix, Corning, NY, USA) was coated onto a 24-well plate according to the recommended protocol, and 500 μL of MCF-7 cell suspension (1.0×10 ⁵ cells/mL) was dropped onto each well and cultured at 37°C and 5% CO ₂ for several days.

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.

Three-dimensional culture of HepG2 cells and evaluation of CYP species activity
250 μL of 0.5 w/v% NFBC-HPC was added to a 24-well plate and allowed to stand. 500 μL of HepG2 cell suspension (1.0 × 10 ⁵ cells/mL) was slowly dropped onto the NFBC-HPC and cultured at 37°C and 5% CO ₂ for 7 days. 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. 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 cells were cultured. At this time, an equal amount of each diluted substrate was also added to blank wells 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 incubated at room temperature for 20 minutes. CYP activity was then evaluated by measuring luminescence intensity using EnSpire (PerkinElmer, MA, USA).

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.

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 suspensions were prepared and added to the NFBC-HPC-containing medium to a concentration of 1.0 × 10 ⁴ cells/mL. The cells were cultured for several days at 37°C in a 5% CO ₂ environment using a Nunc ^TM EasYFlask ^TM 25cm ² (Thermo Fisher Scientific). During the culture, the cells were observed and photographed using a routine inverted microscope Primo Vert (ZEISS).

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 using either cell (Figure 6). Compared to culturing on NFBC-HPC gel, a large number of spheroids could be produced at once by culturing in suspension in a medium containing NFBC-HPC.

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 liquid nitrogen, quickly seeded on SL10 culture dishes, and then cultured at 37°C and 5% _CO2 to plate culture the iPS cells. At the time of subculturing, differentiated iPS cells were removed, 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 volume ratio of 2,000:1:2) was added to the collected iPS cell colonies, and the iPS cell suspension was prepared by pipetting so that the colony size was approximately 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 in a 5% _CO2 environment for several days. During the culture, the plate was observed and photographed using a routine inverted microscope Primo Vert (ZEISS).

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 grew in a culture-day-dependent manner.

Evaluation of undifferentiated state of iPS spheroids
iPS spheroids cultured on NFBC-HPC were harvested, washed with PBS (-), and fixed by adding 1 mL of 4% paraformaldehyde and leaving at room temperature for 15 minutes. 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 rotated at room temperature for 8 hours. The cells were washed three times with PBS (-), and 300 μL of the secondary antibody Alexa Fluor ^TM 488 goat anti-rabbit IgG (H + L) (Thermo Fisher Scientific) diluted 500 times with PBS (-) was added to suspend the cells, and the cells were rotated at room temperature for 1 hour in the dark. The cells were washed with PBS (-), and 200 μL of Hoechst 33342 (DOJINDO) diluted 1,000 times was added to suspend the cells, and the cells were 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.), and a cover glass (Matsunami Glass Industry Co., Ltd.) was placed on top. The cells were observed and photographed using a confocal laser microscope LSM700 (ZEISS).

result
To evaluate the undifferentiated state of iPS spheroids produced 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 produced using NFBC-HPC.

3D cell culture using NFBC-HPC with different dispersant concentrations
The dispersant concentration in the medium during NFBC-HPC preparation 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 left to stand. 500 μL of Colon26 cell suspension (1.0×10 ⁵ cells/mL) was slowly dropped onto the NFBC-HPC. After that, the plates were cultured at 37°C in a 5% CO ₂ environment for 4 days, and the spheroids were observed and photographed using a routine inverted microscope Primo Vert (ZEISS).

Results: 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 on 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.

Suspension culture of cells using NFBC-HPC with different dispersant concentrations
The dispersant concentration in the medium during NFBC-HPC preparation 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 Colon26 cell suspension was prepared and added to the NFBC-HPC-containing medium at 1.0 x ¹⁰⁴ cells/mL. The cells were cultured for 4 days at 37°C and 5% CO2 using _a Nunc ^™ EasYFlask ^{™ 25cm2} (Thermo Fisher Scientific), and observed and photographed using a routine inverted microscope Primo Vert (ZEISS).

ResultsWhen cultured in a NFBC-HPC-containing medium 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 NFBC-HPC-containing medium with a dispersant concentration of 2.0%, most of the spheroids formed were observed to be cultured floating in the medium.

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

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 (12)

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, wherein the bacteria is Gluconacetobacter intermedius SIID9587 strain (accession number NITE P-01495), and the scaffold material has the following physical properties (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) . The scaffold material according to claim 1, wherein the bacterial cellulose is produced by the bacteria 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. 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. The scaffold material according to any one of claims 1 to 3, wherein the culture is a static culture. The scaffold material according to any one of claims 1 to 3, wherein the culture is a suspension culture. The scaffold material according to any one of claims 1 to 3, wherein the culture is a three-dimensional culture. The scaffold material according to any one of claims 1 to 6, wherein the cells are cancer cells. The scaffold material according to any one of claims 1 to 6, wherein the cells are stem cells. The scaffold material according to any one of claims 1 to 6, wherein the cells are primary cultured cells. A medium composition containing the scaffold material according to any one of claims 1 to 9. A coating agent for a cell culture vessel, the coating agent containing the scaffold material according to any one of claims 1 to 9. A method for producing cell spheroids, comprising a step of culturing cells in the presence of a scaffold material according to any one of claims 1 to 9.