WO2018074432A1 - Cell culture substrate and method for producing same, and cell culture container and cell culture method using same - Google Patents

Abstract

[Problem] Provided is a technology which enables the selective formation of spheroids. [Solution] A cell culture substrate containing cellulose fibers, and wherein the arithmetic mean height (Sa) of the culture surface is less than 210 nm.

Description

Cell culture substrate and method for producing the same, and cell culture container and cell culture method using the same

The present invention relates to a cell culture substrate and a method for producing the same, and a cell culture container and a cell culture method using the same.

The cells that form organs such as the liver, pancreas, skin, and blood vessels are three-dimensionally networked in the living body to express their functions.

In research on regenerative medicine that regenerates the functions of these organs, when culturing cells of these organs, a culture in which cells can form a three-dimensional network (that is, three-dimensional culture) is required. . A spheroid is a tissue in which cells form a three-dimensional network. However, when cells are cultured on the surface of a general resin-made cell culture substrate, the cells spread in a plane and proliferate (two-dimensional culture), and a three-dimensional network is not formed.

As a technique for performing three-dimensional culture, Japanese Patent Application Publication No. 2013-54156 (US Patent Application Publication No. 2013/330379, US Patent Application Publication No. 2013/344036, US Patent Application Publication No. 2014/010790). And corresponding to US Pat. No. 9631177) mechanically disrupted fine cellulose fibers (CNF) derived from living cells and plants in the form of hydrogels or membranes forming hydrogels and / or A cell culture matrix containing the derivative is reported. Japanese translations of PCT publication No. 2013-54156 (U.S. Patent Application Publication No. 2013/330379, U.S. Patent Application Publication No. 2013/344036, U.S. Patent Application Publication No. 2014/010790, U.S. Pat. No. 9631177) In the matrix), it is described that cells exist in a three-dimensional arrangement in the matrix.

Japanese translations of PCT publication No. 2013-54156 (U.S. Patent Application Publication No. 2013/330379, U.S. Patent Application Publication No. 2013/344036, U.S. Patent Application Publication No. 2014/010790, U.S. Pat. No. 9631177) In fact, plant-derived CNF hydrogels can be used as biomimetic human extracellular matrix (ECM) for three-dimensional cell culture (paragraphs “0012”, “0080”, “0081”). "). However, Japanese translations of PCT publication No. 2013-54156 (U.S. Patent Application Publication No. 2013/330379, U.S. Patent Application Publication No. 2013/344036, U.S. Patent Application Publication No. 2014/010790, U.S. Pat. No. 9631177). It was difficult to selectively form spheroids even with the technology of No. 1).

Therefore, the present invention has been made in view of the above circumstances, and an object thereof is to provide a technique for performing spheroid-selective cell culture.

Another object of the present invention is to provide a technique for simultaneously realizing cell observability required for performing spheroid-selective cell culture and performing image analysis or the like in an in vitro test.

The present inventors have conducted intensive research to solve the above problems. As a result, the inventors have found that the above problems can be solved by culturing cells on a cell culture substrate containing cellulose fibers and having a specific surface roughness, and have completed the present invention.

That is, the above object can be achieved by a cell culture substrate containing cellulose fibers and having an arithmetic average height (Sa) of the culture surface of less than 210 nm.

FIG. 1 is a photomicrograph showing the growth state of HepG2 cells on the substrate 1, comparative substrates 4 and 5, multiwell cell culture plate and NanoCulture (registered trademark) Plate in the evaluation of HepG2 cell adhesion behavior. FIG. 2 is a photomicrograph showing the growth state of MCF-7 cells on the substrate 1, comparative substrates 4 and 5, multiwell cell culture plate and NanoCulture (registered trademark) Plate in the evaluation of MCF-7 cell adhesion behavior. It is. FIG. 3 is a graph showing the amount of albumin produced on the substrate 1, the multiwell cell culture plate, and the NanoCulture (registered trademark) Plate in HepG2 cell culture. FIG. 4 is a diagram schematically showing the cell culture container of the present invention. In FIG. 4, reference numerals 100, 101 and 102 denote cell culture containers, 1 denotes a cell culture substrate (film), and 20 denotes a support member. FIG. 5 is a photomicrograph showing the growth state of rat primary hepatocytes on the substrate 1 in the evaluation of rat primary hepatocyte adhesion behavior.

One embodiment of the present invention is a cell culture substrate that contains cellulose fibers and has an arithmetic average height (Sa) of a culture surface of less than 210 nm. According to the cell culture substrate according to one embodiment of the present invention, spheroids can be selectively formed.

In the present specification, the cell culture substrate is also simply referred to as “substrate of the present invention” or “substrate”. The arithmetic average height (Sa) is also simply referred to as “surface roughness” or “Sa”.

A substrate conventionally used for culturing adherent cells in vitro is plasma-treated polystyrene. However, when such plasma-treated polystyrene is used, adherent cells grow by attaching to the substrate in a single layer (two-dimensional culture). In the two-dimensional culture, the cell growth is good, but various functions of the cell are lowered, and there is a problem that the function in the living body cannot be reproduced in vitro. In order to solve this problem, in recent years, methods for suppressing cell functional deterioration by forming and culturing cell aggregates (spheroids) have been studied. Japanese translations of PCT publication No. 2013-54156 (U.S. Patent Application Publication No. 2013/330379, U.S. Patent Application Publication No. 2013/344036, U.S. Patent Application Publication No. 2014/010790, U.S. Pat. No. 9631177) When the composition is in the form of a membrane, an opaque membrane is prepared by vacuum filtration of the CNF aqueous dispersion or by drying the filtration membrane under a weight. (Paragraph “0071”). Since the membrane obtained by such a method passes through a filtration membrane used during vacuum filtration, the surface is very rough (Sa is 210 nm or more) and becomes an opaque membrane in which cell observation is difficult. Use in in vitro tests that require observation is difficult. In addition, when cells are cultured on such a CNF membrane, as shown in the following comparative example, in addition to spheroids, cells that grow two-dimensionally (extend) are mixed, or spheroids are not formed, Cells spread in a plane and become a two-dimensional culture. In fact, Japanese translations of PCT publication No. 2013-54156 (U.S. Patent Application Publication No. 2013/330379, U.S. Patent Application Publication No. 2013/344036, U.S. Patent Application Publication No. 2014/010790, U.S. Pat. No. 9631177). In the examples (corresponding to the specification), hydrogels are used for three-dimensional cultures, whereas CNF membranes are used only for two-dimensional cultures (see in particular Example 5 in paragraph "0100").

In contrast, according to the present invention, spheroids are selectively obtained by culturing cells on the culture surface of a cell culture substrate (for example, cellulose fiber membrane) having a specific surface roughness and containing cellulose fibers. Furthermore, the spheroids can be formed selectively and without adhering to the substrate (without floating). The mechanism is still unclear, but is presumed as follows. That is, the base material of the present invention is a cellulose fiber membrane whose culture surface has a specific surface roughness (0 nm <Sa <210 nm). Such a cellulose fiber membrane having moderate irregularities has a balance of appropriate hydrophilicity and hydrophobicity on the surface. For this reason, when cells are cultured on the membrane (culture surface), the cells adhere to the substrate with an appropriate strength due to the unevenness of the culture surface. For this reason, the cells grow three-dimensionally rather than planarly. Therefore, spheroids can be selectively formed by using the substrate of the present invention. On the other hand, if the surface roughness (Sa) of the culture surface is 210 nm or more, the roughness (unevenness) becomes too large and the cell adheres strongly to the base material, so that the cells spread easily and proliferate. In other words, since the seeded cells adhere firmly to the substrate, the cells cannot migrate and cannot form cell aggregates. Therefore, if it culture | cultivates with such a base material, it will become difficult to acquire only a three-dimensional cultured cell.

Therefore, according to the base material of the present invention, the surface of the base material (the culture surface) can be used without requiring pretreatment (for example, a coat of a component that suppresses protein adhesion or a protein coat that promotes or suppresses cell adhesion). ) Can be directly cultured to selectively form spheroids. Moreover, since the cell adheres to the culture surface of the base material without floating with an appropriate adhesive force, the size of the spheroid can be controlled. For this reason, necrosis of the central part of the spheroid, and hence cell functional deterioration can be suppressed / prevented. Therefore, when the base material of the present invention is used, cells (spheroids) having a high function can be obtained. Therefore, the cells (spheroids) cultured using the base material of the present invention can be suitably used for in vitro tests such as regenerative medicine applications, drug efficacy tests during drug development, and toxicity tests.

In addition, as described above, the cells cultured using the base material of the present invention appropriately adhere to the base material. For this reason, even when the medium is exchanged during the cell culture, the cells are rarely removed together with the medium, and therefore the cell loss can be reduced. For example, the culture can be performed for a long period of one week or longer. In addition, since cell aggregation is suppressed, cells of uniform size (spheroids) can be formed, and the function of the cells can be maintained high. In particular, when cells are cultured on a substrate having a haze of 40% or less, cell observation is easy and J. Biomaterial. Sci. Polymer Edn, Vol. 17, No. 8, pp. 859-873 (2006) allows spheroid formation on the membrane without intentional unevenness as described in the document, so that it is possible to clearly observe the morphological changes of cells accompanying various cultures. Become. Thereby, when conducting a drug efficacy test or a toxicity test of a pharmaceutical, it can be suitably used in an in vitro test in which the influence of the pharmaceutical on cell proliferation is measured by a change in spheroid size by image analysis. Therefore, by using such a base material, it is possible to selectively form spheroids and improve the cell observability required when performing image analysis or the like in an in vitro test (cells can be observed more easily) ). Therefore, the cells (spheroids) cultured using the substrate of the present invention can be suitably used for in vitro tests such as regenerative medicine applications, drug efficacy tests during drug development, and toxicity tests.

Furthermore, Japanese translations of PCT publication No. 2013-54156 (U.S. Patent Application Publication No. 2013/330379, U.S. Patent Application Publication No. 2013/344036, U.S. Patent Application Publication No. 2014/010790, U.S. Pat. No. 9631177). In the case of using the hydrogel described in the specification, the water is transported in a state of containing water. However, the risk of contamination of microorganisms due to water adhering to the wall surface or lid of the culture vessel during the transportation process Becomes higher. On the other hand, since the base material of the present invention can be transported in a dry state, there is an advantage that the risk of microbial contamination can be reduced and sterilization such as γ-ray sterilization is easy. Moreover, since the base material of the present invention can be used without pretreatment such as protein coating (a spheroid can be selectively formed), it is possible to reduce the time and labor required for carrying out the test.

In addition, it is known that there are cells that are easily three-dimensional (HepG2, rat primary hepatocytes, etc.) and cells that are difficult to three-dimensional (MCF-7, etc.) depending on the cultured cells. By using the cells, it is possible to easily obtain three-dimensional cultured cells without changing the base material for each cell, as well as cells that are easily three-dimensionalized, as well as cells that are difficult to three-dimensionalize. Furthermore, since spheroids can be formed using cellulose with low cytotoxicity and without using a heterologous protein, safe and highly functional spheroids can be obtained, and can be suitably used for regenerative medicine.

The above mechanism is speculation and does not limit the technical scope of the present invention.

Hereinafter, embodiments of the present invention will be described. In addition, this invention is not limited only to the following embodiment. In this specification, “X to Y” indicating a range includes X and Y, and means “X or more and Y or less”. Unless otherwise specified, measurements such as operation and physical properties are performed under conditions of room temperature (20 to 25 ° C.) / Relative humidity 40 to 50% RH.

The cell culture substrate of the present invention has an arithmetic average height (Sa) of the culture surface of less than 210 nm. Here, when the Sa of the culture surface is 210 nm or more, the surface roughness (unevenness) becomes too large, and the cells spread flatly and easily proliferate, or the seeded cells firmly adhere to the substrate. Therefore, the cells cannot migrate and cannot form cell aggregates. Therefore, it is difficult to selectively acquire only the three-dimensional cultured cells. Considering the further improvement effect of the selectivity of spheroid formation, the arithmetic average height (Sa) of the culture surface is preferably less than 200 nm, more preferably 150 nm or less, and particularly preferably less than 130 nm. The lower limit of the arithmetic average height (Sa) of the culture surface is not particularly limited, but is more than 0 nm, preferably more than 5 nm, more preferably more than 10 nm, still more preferably more than 15 nm. Even more preferably, it is more than 60 nm, particularly preferably 90 nm or more. By culturing on such a culture surface of Sa, spheroids can be further selectively formed and the transparency of the substrate can be further improved (especially haze is further reduced), so that the state of the cells can be more easily visually observed. I can observe. In the present invention, as long as the arithmetic average height (Sa) of at least the culture surface of the substrate is less than 210 nm, the Sa of the other surface is not particularly limited, and may be 210 nm or more. That is, the present invention relates to a form in which the arithmetic average height (Sa) of one surface (culture surface) of the substrate is less than 210 nm and the arithmetic average height (Sa) of the other surface is 210 nm or more, and It includes both forms in which the arithmetic average height (Sa) of both sides of the material is less than 210 nm. Furthermore, Sa is a value at a location where cells adhere substantially, and Sa at a location where cells do not adhere may be any value. That is, the substrate of the present invention only needs to have a Sa of the culture surface at least substantially to which the cells adhere, of less than 210 nm.

In this specification, “arithmetic average height (Sa)” is a parameter obtained by extending the center average roughness (Ra) defined in JIS B0601 (2001) to a plane. In this specification, “arithmetic mean height (Sa)” is a confocal laser microscope (manufactured by Ryoka Systems Co., Ltd.) for a range of 235.3 μm × 470.6 μm of a predetermined surface of a sample (base material or support). The average value of the values measured by the product name: Vert Scan 2.0) is adopted.

As described above, it is preferable that the culture surface of the cell culture substrate exhibits hydrophilicity and hydrophobicity (hydrophobicity) with an appropriate balance. Thereby, spheroid formation on a base material can be promoted more. Here, the hydrophilicity / hydrophobicity of the culture surface is defined by the static water contact angle. Specifically, the static water contact angle of the culture surface of the cell culture substrate is 30 ° or more, preferably 40 ° or more, more preferably more than 45 °. By using a substrate having a culture surface having such a contact angle, spheroid formation on the substrate is further promoted. The upper limit of the static water contact angle is not particularly limited, but is, for example, less than 70 °, preferably less than 65 °, more preferably less than 60 °, and even more preferably less than 55 °. In addition, the said static water contact angle is a value measured by the method described in the following Example.

The cell culture substrate is preferably transparent. Thereby, the cell in culture can be easily observed visually. Here, the transparency can be evaluated by total light transmittance and haze (turbidity). For example, the total light transmittance is preferably 80% or more, more preferably 85% or more (upper limit: 100%). The haze (turbidity) is preferably 40% or less. That is, according to a preferred embodiment of the present invention, the haze of the cell culture substrate is 40% or less. The haze of the cell culture substrate is more preferably 20% or less, still more preferably less than 16%, still more preferably 12% or less, and particularly preferably less than 10%. With such a total light transmittance and / or haze (turbidity), cells in culture can be more easily visually observed. In addition, since the haze of the cell culture substrate is preferably as low as possible, the lower limit is not particularly limited, but is usually 0.1% or more, and 1% or more is sufficient. In addition, the said total light transmittance or haze is a value measured by the method described in the following Example.

In the present invention, the cell culture substrate is a membrane containing cellulose fibers, preferably a membrane composed only of cellulose fibers. Since the cellulose fiber has high safety, for example, it is safe to be implanted in the body. In addition, when cells cultured on cellulose fibers are transplanted into the body, safety can be ensured even if trace amounts of cellulose fibers are included. By culturing using the cell culture substrate of the present invention, the cells can be applied to the body together with the substrate. Here, the average diameter (diameter) of the cellulose fiber is not particularly limited, but is, for example, 2 to 20 nm. Since the base material produced using such a cellulose fiber is excellent in transparency and has a low haze, the visibility of cells during culture can be further improved. In this specification, the value measured according to the following method is employ | adopted for the average diameter (diameter) and average length of a cellulose fiber.

(Measuring method of average diameter (diameter) and average length of cellulose fiber)
An aqueous solution containing 1.0% by weight of cellulose fibers is applied on an acrylic plate so that the film thickness (dry film thickness) is 20 to 50 nm and dried to prepare a coating film. Next, the obtained coating film was observed with an electron microscope (SEM) image at a magnification of 2000 times, and the diameter and length of the cellulose fibers existing in the field of width: 1280 pixels and length: 800 pixels were measured. And the obtained values are taken as the average diameter (diameter) and average length of the cellulose fibers, respectively. In addition, when the cross section of a cellulose fiber is an indeterminate form, let the maximum diameter be the said diameter. In addition, in this specification, a cellulose fiber means the elongate cellulose whose aspect-ratio (average length / average diameter (average diameter)) exceeds 1.

Considering the transparency of the substrate, the content of thick cellulose fibers having a diameter of 1 μm or more is preferably small. That is, according to the preferable form of this invention, the content rate of the cellulose fiber which has a diameter of 1 micrometer or more (with respect to a base material) is less than 10%. More preferably, the content of cellulose fibers having a diameter of 1 μm or more is more preferably 5% or less, and particularly preferably less than 1% (lower limit: 0%). If the content of thick cellulose fibers is in the above range, the substrate is further excellent in transparency (especially the haze of the substrate can be significantly reduced), and cells in culture can be more easily visually observed. When conducting a test or toxicity test, it can be suitably used in an in vitro test in which the influence of a pharmaceutical product on cell proliferation is measured by a change in the size of a spheroid by image analysis. In this specification, the value measured according to the following method is employ | adopted for the content rate of the cellulose fiber whose diameter is 1 micrometer or more.

(Measurement method of content of cellulose fiber having a diameter of 1 μm or more)
Each substrate (size: 1.5 cm × 1.5 cm, area: Y (cm ² )) was imaged with a scanning electron microscope (magnification: 2000 times), and the obtained image was image analysis software (WinROOF2015 (Mitani) Trading Co., Ltd.) is used to determine the area (X (cm ² )) occupied by fibers having a thickness (diameter) of 1 μm or more under the following conditions. The area (X (cm ² )) is divided by the substrate area (Y (cm ² )), and the ratio of the area of the fibers having a thickness (diameter) of 1 μm or more in the substrate area [= (X / Y ) × 100 (%)] is calculated, and this is defined as the content (%) of cellulose fibers having a diameter of 1 μm or more. In the present specification, the content (%) of cellulose fibers having a diameter of 1 μm or more is also referred to as “thick fiber content (%)”. Further, the area of the fiber having a thickness (diameter) of 1 μm or more is also referred to as “area occupied by the thick fiber (cm ² )”. In addition, when the thickness of a fiber (cellulose fiber) is not uniform, when the thickness (diameter) is 1 μm or more over 60% or more of the total length of the fiber, the fiber is a fiber having a thickness (diameter) of 1 μm or more. I reckon. Further, when the cross section of the fiber is irregular, the maximum diameter is regarded as “thickness (diameter)”.

The thickness of the cell culture substrate is not particularly limited, but the thickness (dry film thickness) of the cell culture substrate is preferably 10 to 100 μm, more preferably 20 to 60 μm.

As described above, the base material of the present invention is characterized in that at least a surface to be a culture surface has a specific surface roughness (Sa). The method for forming such a surface is not particularly limited, but is preferably controlled by the surface roughness of the coated surface of the support on which the cellulose fiber dispersion is coated. That is, when a coating solution containing cellulose is applied to a support and dried to form a coating film, the coating surface on the support surface side is affected by the surface roughness of the coating surface of the support. For this reason, the present inventors thought that the base material of the present invention could be easily obtained by appropriately adjusting the surface roughness of the support. As a result of further intensive studies based on the above estimation, the use of a support having an arithmetic average height (Sa) of the surface (application surface) on which cellulose fibers are applied (less than 30 nm) of the present invention. It has been found that a substrate can be easily obtained. Therefore, this invention also provides the manufacturing method of the base material for cell cultures which has apply | coating the coating liquid containing a cellulose fiber on the support body whose arithmetic mean height (Sa) of a culture surface is less than 30 nm. .

Hereinafter, a preferable method for producing the cell culture substrate will be described. The following description does not exclude that the cell culture substrate of the present invention is produced by other methods.

First, a coating liquid containing cellulose fibers (hereinafter also simply referred to as “coating liquid”) is prepared.

Here, the production method of the cellulose fiber is not particularly limited, and a method of mechanically defibrating cellulose (cellulose fiber raw material) can be used. For the production of cellulose fibers, for example, known methods such as those described in JP-A-2016-87877, JP-A-2015-218299, JP-A-2015-140403 and the like can be used similarly or appropriately. Can be used with modification. Hereinafter, although the preferable form of the manufacturing method of a cellulose fiber is demonstrated, this invention is not limited to the following form.

Cellulose (cellulose fiber raw material) is not particularly limited, and may be plant-derived or bacterial-derived cellulose. From the viewpoint of availability, cost, etc., plant-derived cellulose may be used as the cellulose fiber raw material. preferable. For example, various woods obtained from larch, cedar, oil palm, cypress, etc .; pulps; papers such as newspapers, cardboard, magazines, fine papers; plant shells such as rice husks, palm husks, coconut husks, etc. May be. Here, the pulp is selected from wood pulp, non-wood pulp, and deinked pulp. Here, the wood pulp is not limited to the following, but hardwood bleached kraft pulp, hardwood unbleached kraft pulp, conifer bleached kraft pulp, softwood unbleached kraft pulp, sulfite wood pulp, soda pulp, unbleached kraft pulp, oxygen bleach Chemically modified pulp such as kraft pulp and hydrolyzed kraft pulp; Semi-chemical pulp such as semi-chemical pulp (SCP) and Chemi-ground wood pulp (CGP); such as groundwood pulp (GP) and thermomechanical pulp (TMP, BCTMP) Examples include mechanical pulp. Non-wood pulp is not limited to the following, but cotton pulp such as cotton linter and cotton lint (cotton cellulose), hemp (hemp cellulose), straw (straw cellulose), bagasse (sugar cane squeezed), empty Non-wood pulp such as fruit bunches (EFB), rice straw, corn stalks, cellulose isolated from sea squirts, seaweeds, and the like. The deinking pulp is not limited to the following, and examples include deinking pulp made from waste paper. The said cellulose fiber raw material may be used individually by 1 type, or may be used with the form of a 2 or more types of mixture. Among these, wood pulp and chemically modified pulp containing cellulose are preferable from the viewpoints of availability, ease of control of fiber diameter, fiber refinement (defibration), and the like. Alternatively, commercially available cellulose fiber raw materials may be used.

The cellulose fiber raw material may be subjected to physical or chemical treatment in advance before mechanical defibration. Specifically, degreasing treatment, delignification treatment (holocellulose conversion), alkali treatment, oxidation treatment and the like can be mentioned. Two or more of the above treatments may be used in combination.

Here, the degreasing treatment method is not particularly limited, and a known method can be used. Specifically, it can be performed by immersing the cellulose fiber raw material in a degreasing solution. Here, the solvent used for the preparation of the degreasing solution is not particularly limited, and may be appropriately selected depending on the type of the cellulose fiber raw material to be used. For example, water, acetone, alcohol, etc. are mentioned. The said solvent may be used individually by 1 type, or may be used with the form of 2 or more types of mixed solutions. The degreasing conditions are not particularly limited and may be appropriately selected depending on the type of cellulose fiber raw material to be used. For example, the degreasing temperature is usually 10 to 100 ° C., preferably 15 to 50 ° C. The degreasing time is usually 1 to 30 hours, preferably 15 to 20 hours. Moreover, the said degreasing process may be performed under stirring.

The delignification (holocellulose) method is not particularly limited, and a known method can be used. Specifically, the cellulose fiber raw material is added to a delignification solution containing an acid (eg, sulfuric acid, hydrochloric acid, acetic acid, acetic anhydride) and an oxidizing agent (bleaching agent) (eg, sodium chlorite, hydrogen peroxide). And the method of heating this can be used preferably. Here, the heating conditions are not particularly limited, and may be appropriately selected depending on the type of cellulose fiber raw material used and the type of acid or oxidizing agent. For example, the heating temperature is usually 50 to 120 ° C., preferably 60 to 100 ° C. The heating time is usually 0.5 to 5 hours, preferably 1 to 3 hours.

The alkali treatment method is not particularly limited, and a known method can be used. Specifically, it can be performed by immersing the cellulose fiber raw material in an alkaline solution. Here, the alkali used for preparing the alkaline solution is not particularly limited, and may be inorganic or organic. For example, as an inorganic substance (inorganic alkali), lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium carbonate, lithium hydrogen carbonate, potassium carbonate, potassium hydrogen carbonate, sodium carbonate, sodium hydrogen carbonate, calcium carbonate, Examples thereof include hydroxides, carbonates and phosphates of alkali metals or alkaline earth metals such as lithium phosphate, potassium phosphate, trisodium phosphate, disodium hydrogen phosphate, calcium phosphate, and calcium hydrogen phosphate. It is done. Examples of the organic substance (organic alkali) include ammonia; hydrazine, methylamine, ethylamine, diethylamine, triethylamine, propylamine, dipropylamine, butylamine, diaminoethane, diaminopropane, diaminobutane, diaminopentane, diaminohexane, cyclohexyl. Amine, aniline, tetramethylammonium hydroxide, tetraethylammonium hydroxide, tetrapropylammonium hydroxide, tetrabutylammonium hydroxide, benzyltrimethylammonium hydroxide, pyridine, N, N-dimethyl-4-aminopyridine, ammonium carbonate, carbonic acid Aliphatic amines, aromatic amines, aliphatic ammoniums, fragrances such as ammonium hydrogen and diammonium hydrogen phosphate Ammonium, heterocyclic compounds, and their hydroxides, and carbonates, and phosphate. Moreover, the solvent used for preparation of an alkaline solution will not be specifically limited if an alkali can be melt | dissolved, Lower alcohols, such as water, methanol, and ethanol, can be used. Preferably, it contains water, that is, an aqueous sodium hydroxide solution and an aqueous potassium hydroxide solution can be preferably used as the alkaline solution. Here, the alkali concentration in the alkali solution is not particularly limited, but is, for example, 1 to 15% by weight, preferably 3 to 7% by weight. The alkali treatment conditions are not particularly limited, and may be appropriately selected depending on the cellulose raw material to be used and the type of alkali. For example, the alkali treatment temperature is usually 10 to 50 ° C., preferably 15 to 40 ° C. The alkali treatment time is usually 0.5 to 5 hours, preferably 1 to 3 hours. Moreover, the said alkali treatment may be performed under stirring. In addition, when performing an alkali treatment, you may wash | clean the cellulose after an alkali treatment and remove an excess alkali. Here, the cleaning liquid that can be used for cleaning is not particularly limited, but the same solvent as that used for the alkaline solution can be used.

The oxidation treatment method is not particularly limited, and a known method can be used. Specifically, there is a method using an N-oxyl compound as an oxidation catalyst. Thereby, the cellulose surface can be selectively oxidized and the cellulose can be easily refined. Moreover, the oxidation reaction can be carried out in an aqueous system and under relatively mild conditions (normal pressure near room temperature). In addition, the oxidation reaction of cellulose in wood also proceeds selectively on the crystal surface from the inside of the crystal, and the alcoholic primary carbon possessed by the cellulose molecular chain can be selectively converted into a carboxyl group. For this reason, cellulose fibers can be dispersed one by one in an aqueous solvent by mechanical defibration in the next step. Therefore, the aqueous dispersion of cellulose fibers obtained by this embodiment has high transparency. For this reason, from the viewpoint of further improving the transparency of the substrate, it is preferable to use oxidized (carboxylated) cellulose as a cellulose fiber raw material. Here, the N-oxyl compound is not particularly limited, and a known catalyst used for the oxidation of cellulose can be used. Specifically, 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), 2,2,6,6-tetramethyl-4-hydroxypiperidine-1-oxyl, 4-methoxy-2,2 , 6,6-Tetramethylpiperidine-N-oxyl, 4-ethoxy-2,2,6,6-tetramethylpiperidine-N-oxyl, 4-acetamido-2,2,6,6-tetramethylpiperidine-N -Oxyl, 4-carboxy-2,2,6,6-tetramethylpiperidine-N-oxyl, 4-phosphonooxy-2,2,6,6-tetramethylpiperidine-N-oxyl and the like. Of these, TEMPO and 4-acetamido-2,2,6,6-tetramethylpiperidine-N-oxyl are preferable and TEMPO is more preferable from the viewpoint of the oxidation efficiency of cellulose. The amount of the N-oxyl compound used is not particularly limited as long as it is an amount capable of proceeding with the cellulose oxidation. For example, the amount of the catalyst used is preferably about 0.1 to 5% by weight, more preferably about 0.5 to 3% by weight, based on the cellulose fiber raw material.

The oxidation treatment may be performed in the presence of an oxidizing agent. By using an oxidizing agent in combination, the introduction efficiency of the carboxy group can be further improved. Moreover, since the oxidation reaction can be performed under mild conditions, it is easy to maintain the crystal structure of cellulose. Here, the oxidizing agent is not particularly limited. For example, halogen (chlorine, bromine, iodine, etc.), hypohalous acid or a salt thereof (hypochlorous acid or a salt thereof, hypobromous acid or a salt thereof, Hypoiodic acid or a salt thereof), halogenous acid or a salt thereof (chlorous acid or a salt thereof, bromous acid or a salt thereof, iodic acid or a salt thereof), a perhalogenic acid or a salt thereof (peroxide) Chloric acid or its salt, periodic acid or its salt, etc.), halogen oxide (ClO, ClO ₂ , Cl ₂ O ₆ , BrO ₂ , Br ₃ O ₇ etc.), nitrogen oxide (NO, NO ₂ , N ₂₎ O ₂ ), peroxides (hydrogen peroxide, peracetic acid, persulfuric acid, perbenzoic acid, etc.). From the viewpoint of the oxidation efficiency of cellulose and the like, hypohalous acid and hypohalite are preferable, and sodium hypochlorite (NaClO) is more preferable. The said oxidizing agent may be used independently or may use 2 or more types together. Further, the oxidizing agent may be added as it is or in the form of a solution dissolved in an appropriate solvent (for example, water). The amount of the oxidizing agent to be used is not particularly limited as long as it can promote the oxidation reaction. For example, it is preferably about 1 to 30 mmol, more preferably about 5 to 20 mmol with respect to 1 g of the cellulose fiber raw material.

Further, when the oxidation treatment is performed in the presence of the oxidant, the oxidation treatment may be further performed in the presence of bromide and / or iodide. As a result, the oxidation reaction can proceed more smoothly. Here, examples of the bromide include, but are not limited to, ammonium bromide, sodium bromide, lithium bromide and the like. Similarly, examples of iodide include, but are not limited to, ammonium iodide, sodium iodide, lithium iodide, and the like. Of these, sodium bromide (NaBr) is preferable from the viewpoint of cost and stability. The bromide and iodide may be used alone or in combination of two or more. In addition, bromide and iodide may be used in combination. The amount of bromide and / or iodide used is not particularly limited as long as it can promote the oxidation reaction. For example, it is preferably 0.5 to 30% by weight, more preferably 1 to 10%, based on the cellulose fiber raw material. It is about wt%.

The oxidation treatment conditions are not particularly limited, and may be appropriately selected depending on the cellulose fiber raw material to be used, the oxidation catalyst, and the oxidizing agent when used and the type of bromide / iodide (when used). For example, the oxidation treatment temperature is usually 10 to 50 ° C., preferably 15 to 40 ° C. The oxidation treatment time is usually 0.5 to 5 hours, preferably 1 to 3 hours. The oxidation treatment may be performed with stirring.

In addition, after the oxidation, a reduction reaction may be performed if necessary. Here, the reducing agent is not particularly limited and a known reducing agent such as sodium borohydride (NaBH ₄₎ can be used. Here, the amount of the reducing agent to be used is not particularly limited as long as the reduction can proceed to a desired level. For example, the amount of the reducing agent is preferably 1 to 30% by weight, more preferably with respect to the initial charge amount of the cellulose fiber material. Is about 5 to 20% by weight. The reduction reaction conditions are not particularly limited, and may be appropriately selected depending on the cellulose fiber raw material to be used and the type of the reducing agent. For example, the reduction reaction temperature is usually 10 to 50 ° C., preferably 15 to 40 ° C. The oxidation treatment time is usually 0.5 to 5 hours, preferably 1 to 3 hours. Moreover, the said reduction process may be performed under stirring.

By the above reaction, oxidized cellulose having a carboxylate content of 0.1 to 3.0 mmol / g, preferably 0.2 to 2.0 mmol / g is obtained. Oxidized cellulose having such a carboxylate content can be finely divided and uniformly dispersed by electrostatic repulsion between celluloses.

The cellulose obtained as described above is mechanically defibrated. Thereby, a cellulose further refines | miniaturizes and a cellulose fiber is obtained. Here, the method for mechanical fibrillation (miniaturization) of cellulose is not particularly limited, and a known method can be used. Specifically, a method of refining (defibrating) an aqueous dispersion of cellulose using a defibrating apparatus can be used. Here, examples of the aqueous medium used for obtaining the aqueous dispersion include water and lower alcohols (methanol, ethanol, propanol, isopropanol) and the like. The aqueous medium may be used alone or in the form of a mixture of two or more. Of these, water is preferred. Here, the cellulose concentration in the aqueous dispersion is not particularly limited, but is preferably about 0.1 to 20% by weight, more preferably about 0.3 to 10% by weight, from the viewpoint of mechanical defibration (miniaturization) efficiency and the like. It is. If necessary, the pH of the dispersion may be adjusted to improve the dispersibility of cellulose. Next, the aqueous dispersion is subjected to a mechanical defibrating treatment to refine the cellulose. Here, the mechanical defibrating treatment is not limited to the following, but is a high pressure homogenizer, an ultra high pressure homogenizer, a ball mill, a roll mill, a bead mill, a cutter mill, a planetary mill, a jet mill, an attritor, a grinder, a juicer mixer, a homomixer, an ultra mixer. Examples include mechanical processing such as a sonic homogenizer, a nanogenizer, an underwater collision, a disk refiner, and a conical refiner. In addition, a wet pulverizing apparatus such as a twin-screw kneader, a vibration mill, a homomixer under high-speed rotation, an ultrasonic disperser, and a beater can be used as appropriate. By performing such mechanical fibrillation treatment, the cellulose in the dispersion is refined, and a cellulose fiber dispersion can be obtained.

The cellulose fiber dispersion thus obtained may be used as it is as a coating liquid containing cellulose fibers (coating liquid). Alternatively, the cellulose fibers may be separated from the cellulose fiber dispersion obtained as described above, and then dispersed in an appropriate solvent to form a coating solution. The solvent that can be used in the latter case is not particularly limited, but is preferably a solvent that does not adversely affect cell culture. Considering the above points, for example, water, lower alcohols (methanol, ethanol, propanol, isopropanol) and the like can be mentioned. The said solvent may be used independently or may be used with the form of a 2 or more types of liquid mixture. Of these, water is preferred. Here, the cellulose fiber concentration in the coating solution is not particularly limited, but is preferably about 0.1 to 20% by weight, more preferably about 0.5 to 10% by weight, from the viewpoints of ease of coating, easy visual observation, and the like. It is. You may concentrate the cellulose fiber dispersion liquid obtained above so that it may become the said density | concentration range. If necessary, the pH of the dispersion may be adjusted to improve the dispersibility of cellulose.

The coating solution may contain only cellulose fibers (that is, the substrate may be composed only of cellulose fibers) or may contain other components (that is, the substrate may contain other components in addition to the cellulose fibers). ) Good. Other components in the latter case are not particularly limited, but include components used in cell culture (eg, serum, various growth factors, differentiation-inducing factors, antibiotics, hormones, amino acids, sugars, salts, etc.) Can be mentioned. In addition, when the coating solution contains other components, the amount of other components added is not particularly limited as long as it does not adversely affect the cultured cells. For example, 0.01 to 100 with respect to cellulose fibers. % By weight.

Next, the coating solution prepared as described above is applied to the surface (culture surface or coated surface) where Sa of the support is less than 30 nm. Here, when the coating liquid containing cellulose fibers is applied to the support surface having an arithmetic average height (Sa) of 30 nm or more, the arithmetic average height (Sa) on the support side of the obtained base material is 210 nm or more ( See Comparative Example 1 below). The arithmetic average height (Sa) of the cellulose fiber-coated surface of the support is preferably 5 nm or more from the viewpoint of obtaining a substrate that can form spheroids more selectively. Among these, it is more preferably 5 to 25 nm, still more preferably 8 to 20 nm, and particularly preferably 10 to 20 nm. By using a support having such a surface of Sa, Sa on the culture surface of the substrate can be controlled within the preferred range as described above. In addition, as long as the arithmetic average height (Sa) of at least the coated surface (cotton to which the coating solution is coated) of the support is less than 30 nm, Sa on the other surface is not particularly limited, and may be 30 nm or more. That is, the present invention provides an embodiment in which the arithmetic average height (Sa) of one surface (application surface) of the support is less than 30 nm and the arithmetic average height (Sa) of the other surface is 30 nm or more, and the support It includes both forms in which the arithmetic mean height (Sa) of both sides of the body is less than 30 nm.

The material of the support is not particularly limited as long as it can form a surface with Sa of less than 30 nm. Specifically, inorganic glass; carbon; metal such as silicon; polyolefin resin such as polyethylene, polypropylene and cyclic olefin; polyester resin such as polyethylene terephthalate (PET); acrylic resin such as polymethyl methacrylate; epoxy resin; Vinyl chloride, polyvinylidene chloride, polystyrene resin, polyvinyl acetate, ABS (acrylonitrile-butadiene-styrene) resin, polycarbonate resin, vinyl ether, polyacetal, polyphenylene ether (PPE), polyaryl ether, polyphenylene sulfide (PPS), polyether Ether ketone (PEEK), polyaryl ether ketone, phenol resin, polyether nitrile (PEN), polyamide resin, polyimide resin, fluorinated poly Bromide resin (fluorine-containing polyimide resin), fluorine resin, polysulfone, polyethersulfone, polydimethylsiloxane and the like. The thickness of the support is not particularly limited, but is usually 1 to 10 mm, preferably 1.5 mm or more and less than 5 mm. In addition, even if a support having a surface roughness of a predetermined Sa value is purchased, the surface of the support is polished by a polishing machine or the like so that the Sa value of the support surface becomes a predetermined value. Also good.

Application method is not particularly limited, natural coater, knife belt coater, floating knife, roll coat, air knife coat, knife over roll, knife on blanket, spray, dip, kiss roll, squeeze roll, reverse roll, air blade, Various coating methods using apparatuses such as a curtain flow coater, a doctor blade, a wire bar, a die coater, a comma coater, a spin coater, an applicator, a baker applicator, a gravure coater, and a screen printing machine may be mentioned. The application may be repeated several times to dozens of times. Further, the amount of the coating solution applied to the support is not particularly limited, but it is preferably an amount that provides the thickness of the substrate as described above. In addition, as described above, as long as the arithmetic average height (Sa) of at least the culture surface of the substrate is less than 210 nm, the Sa of the other surface is not particularly limited, and may be 210 nm or more. For this reason, you may further laminate | stack the film made from a cellulose fiber whose arithmetic mean height (Sa) is 210 nm or more on the coating liquid on a support body.

The drying conditions after application are not particularly limited, and can be appropriately selected mainly considering the boiling point of the solvent. For example, the drying temperature is preferably 20 to 100 ° C., more preferably 40 to 70 ° C. The drying time is preferably 10 to 30 hours, preferably 15 to 20 hours.

The cell culture substrate of the present invention can be obtained by peeling the coating film formed by such a method from the support.

In the above-described method, when a substrate is prepared by applying and drying a coating solution on a surface having a specific roughness (Sa) of the support, one surface of the substrate (ie, the surface of the support) Only the coated surface) has the surface roughness (Sa) defined in the present invention. On the other hand, after coating the coating liquid on the surface having the specific roughness (Sa) of the support, the support having the specific roughness (Sa) is placed on the coating surface (where no support is installed). By placing it, a base material having both surface roughness (Sa) defined in the present invention can be produced.

As described above, the substrate for cell culture according to the present invention is a membrane containing cellulose fibers having an arithmetic average height (Sa) of less than 210 nm on the culture surface, in particular, a portion to which cells substantially adhere. According to the substrate of the present invention, a state in which spheroids are selectively and directly attached to the substrate surface (culture surface) without the need for pretreatment (for example, a coat of a component that suppresses protein adhesion). Can be formed. For this reason, when the base material of the present invention is used, a highly functional cell mass (spheroid) is obtained.

The cell culture substrate of the present invention is preferably sterilized before use for culturing and then used for culturing. As the sterilization treatment for cell culture instruments, known sterilization methods such as autoclave sterilization, dry heat sterilization, ethylene oxide gas sterilization, gamma ray sterilization, and electron beam sterilization can be used. Among these, gamma ray sterilization is preferable because it can be applied to sterilization of instruments having low heat and pressure resistance and there is no problem of residual gas such as ethylene oxide gas.

It is known that the amount of protein attached to the substrate and the adhesion / form of cells are closely related, and the amount of protein adsorbed to the substrate is also controlled in the cell culture substrate of the present invention. Thus, a three-dimensional cultured cell can be obtained more suitably. Specifically, the amount of albumin adsorbed on the substrate is preferably 1000 ng / cm ² or more. Although the upper limit of the amount of adsorption of albumin is not particularly limited, from the viewpoint of formation of spheroids is better, preferably it is preferably 2000 ng / cm ² or less, more preferably 1500 ng / cm ² or less . Instead of or in addition to the above-mentioned albumin adsorption amount, the substrate proteoglycan adsorption amount is preferably 160 ng / cm ² or more. The upper limit of the adsorption amount of proteoglycan is not particularly limited, but is preferably 300 ng / cm ² or less, more preferably 200 ng / cm ² or less, from the viewpoint of better spheroid formation. The adsorption amount of albumin and the adsorption amount of proteoglycan are values measured by the methods described in the following examples.

Therefore, the cell culture substrate of the present invention can be suitably used for a culture vessel. That is, in one embodiment of the present invention, a culture container having the cell culture substrate of the present invention is provided. As long as cells are cultured on the base material of the present invention, the culture container (cell culture container) of the present invention may be configured by combining the cell culture base material of the present invention and other members, The cell culture substrate of the present invention and the other member may be integrated, or may be composed only of the cell culture substrate of the present invention. When the cell culture substrate of the present invention is a flexible substrate such as a film, it may be formed in combination with an appropriate support member having rigidity.

FIG. 4 illustrates one embodiment of the cell culture container according to the present invention. The cell culture vessel may be composed of the cell culture substrate 1 as shown in FIG. 4 (A), or the cell culture substrate 1 and the support member 20 as shown in FIGS. 4 (B) and (C). It may consist of. In FIG. 4, the inner shape and the outer shape when the cell culture container is viewed in plan from the opened side can be any shape such as a circle, a polygon (square, triangle, etc.), for example. Examples of the material constituting the support member 20 include inorganic glass; carbon; metal such as silicon; polyolefin resin such as polyethylene, polypropylene and cyclic olefin; polyester resin such as polyethylene terephthalate (PET); acrylic such as polymethyl methacrylate. Resin; epoxy resin; polyvinyl chloride, polyvinylidene chloride, polystyrene resin, polyvinyl acetate, ABS (acrylonitrile-butadiene-styrene) resin, polycarbonate resin, vinyl ether, polyacetal, polyphenylene ether (PPE), polyaryl ether, polyphenylenesulfur Examples include fido (PPS), polyether ether ketone (PEEK), polyaryl ether ketone, phenol resin, polyether nitrile (PEN), and the like.Or you may use the said support body as a supporting member.

The cell culture container of the present invention may have any shape as a whole as long as it includes the cell culture substrate of the present invention. For example, it can be in the form of various containers such as plates for culture such as single or multiwell plates, petri dishes, dishes, flasks, bags and the like. The cell culture container of the present invention may also be in the form of a cell culture container in a culture apparatus such as a mass culture apparatus or a perfusion culture apparatus.

In one embodiment of the present invention, there is provided a cell culture method comprising culturing cells (adherent cells) on the culture surface of the cell culture substrate of the present invention. The cell culture substrate can selectively form spheroids by culturing cells on the culture surface according to the surface roughness (Sa). In addition, since the cells adhere to the culture surface of the substrate with an appropriate adhesive force, the size of the spheroid can be controlled. For this reason, necrosis of the central part, and hence the decrease in cell function can be suppressed / prevented. In addition, since the cells cultured using the substrate of the present invention adhere appropriately to the substrate, even when the medium is changed during cell culture, the cells are rarely removed together with the medium. Loss can be reduced. In addition, since cell aggregation is suppressed, cells of uniform size (spheroids) can be formed, and the function of the cells can be maintained high. In a further embodiment of the present invention, a cell culture method is provided in which cells (particularly adherent cells) are three-dimensionally cultured in the culturing step. That is, in one embodiment of the present invention, the culture is three-dimensional culture.

The type of cells cultured by the cell culture method of the present invention is not particularly limited, and normal cells, cancer cells, stem cells, and fused cells such as hybridomas can be used, and cells that have been subjected to artificial treatment such as gene transfer It may be. Although not particularly limited, for example, three-dimensional culture of artificial pluripotent stem cells (Induced pluripotent stem cells: iPS cells), embryonic stem cells (Embryonic stem cells: ES cells), mesenchymal stem cells, etc. may be generally performed. Adipose cells, hepatocytes, kidney cells, pancreatic cells, mammary cells, endothelial cells, epithelial cells, smooth muscle cells, myoblasts, cardiomyocytes, nerve cells, including the required cells and various progenitor cells and stem cells, Glial cells, dendritic cells, chondrocytes, osteoblasts, osteoclasts, bone cells, fibroblasts, various blood cells, retinal cells, cornea-derived cells, gonad-derived cells, various line cells, other mesenchymal precursors And cells such as cells and various cancer cells. The biological species from which these cells are derived is not particularly limited, and various cells derived from humans and non-human animals can be used. Examples of the biological species from which the cells are derived include, for example, primates such as humans, rhesus monkeys, green monkeys, cynomolgus monkeys, chimpanzees, tamarins and marmosets, rodents such as mice, rats, hamsters and guinea pigs, dogs, cats, rabbits, pigs, Examples include cows, goats, sheep, horses, chickens, quails, minks, pineapples, and zebrafish.

The medium used for cell culture may be appropriately selected according to the cells. The type of medium is not particularly limited. For example, any cell culture basic medium, differentiation medium, primary culture medium, or the like can be used. Specifically, Eagle Minimum Essential Medium (EMEM), Dulbecco's Modified Eagle Medium (DMEM), α-MEM, Glasgow MEM (GMEM), IMDM, RPMI 1640, Ham F-12, MCDB Medium, Williams Medium E, and these Examples thereof include, but are not limited to, a mixed medium, and any medium can be used as long as the medium contains components necessary for cell growth and differentiation. Furthermore, a medium supplemented with serum, various growth factors, differentiation-inducing factors, antibiotics, hormones, amino acids, sugars, salts and the like may be used. The culture temperature is not particularly limited, but is usually about 25 to 40 ° C.

Examples of tissues formed by three-dimensional culture include spheroids and three-dimensional cell aggregates. A spheroid or a three-dimensional cell aggregate is a spheroid or a three-dimensional cell aggregate formed of a single cell such as a hepatocyte. It may be a spheroid mixed with cell types or a three-dimensional cell aggregate. Examples of cells that can be used include the various cells described above. In one embodiment, there is provided the cell culture substrate, wherein the cell culture substrate has a spheroid selectively adhered to the cell culture surface. In another embodiment, a cell culture container having a cell culture substrate having spheroids selectively adhered to the cell culture surface is provided.

By using the substrate of the present invention, a three-dimensional cultured cell having a function close to that of a living body can be obtained. Although the reason is not clear, as shown in the following [Albumin quantification], the three-dimensional cultured cells cultured on the base material of the present invention can exhibit higher functions than conventional culture plates. It is different from three-dimensional cultured cells cultured on a substrate. Therefore, the present invention also provides a three-dimensional cultured cell formed (cultured) on the cell culture substrate of the present invention. It is known that when a three-dimensional cultured cell (spheroid) has a diameter exceeding 200 μm in diameter, nutrients and oxygen in the medium do not reach the central part of the spheroid, and the cells in the central part are necrotic. By forming a spheroid using a material, a large number of spheroids having a diameter of 200 μm or less can be easily obtained. Therefore, the function of the cultured cells can be improved by forming spheroids using the substrate of the present invention. In addition, although the usefulness of the three-dimensional cultured cell formed on the cell culture substrate according to the present invention is as shown in the Examples, it is compared with the three-dimensional cultured cell cultured on the conventional culture substrate. However, it is very difficult to specify the feature points on the structure and characteristics.

It is also possible to carry out drug efficacy tests and toxicity tests at the time of drug development using three-dimensional cultured cells obtained using the cell culture substrate of the present invention. That is, the present invention also provides a method for testing a drug in vitro, comprising using the three-dimensional cultured cell of the present invention. Specifically, in the development of anticancer agents, it is required to screen anticancer agents effective for high throughput by conducting in vitro drug efficacy tests on cancer cells. Since the acquired cancer cells (three-dimensional cultured cells) have resistance to an anticancer agent close to that in the living body, it is possible to acquire drug efficacy data of the anticancer agent that is close to the test performed in the living body. In addition, using three-dimensional cultured cells obtained using the cell culture substrate described in this patent, PLoS One. 2013 Oct 24: 8 As described in 8 (10), it is possible to obtain dose response curve data for measuring the survival rate of cancer cells at each anticancer drug concentration by changing the anticancer drug concentration as needed. Drug efficacy data can be obtained. As a method for measuring the survival rate, a known method can be used. Specifically, a method such as MultiTox-Fluor Multiplex Cytotoxicity Assay kit (Promega) for measuring the survival rate as an index, RubyGlowTM A method using ATP as an index, such as Luminescent Cell Viability Assay Kit (Cosmo Bio Inc.), a method of nucleic acid staining with a fluorescent reagent using Cytotoxic Fluoro-test wako (Wako Pure Chemical Industries), trypan blue using trypan blue Methods for measuring cell viability such as dye exclusion test methods can be used. Furthermore, by measuring changes in the diameter of spheroids after addition of an anticancer agent using an imaging device such as cell3 imager (SCREEN Holdings Co., Ltd.), it is also possible to measure the cell growth inhibitory effect of the anticancer agent. Use imaging devices to evaluate the efficacy of anticancer drugs at higher throughput than methods that measure the viability of other cells that measure the viability using protease activity as an index, such as Fluor Multiplex Cytotoxicity Assay Kit (Promega). This method is more preferable. In particular, when the substrate of the present invention is excellent in transparency, cell function evaluation by image analysis using an imaging apparatus or the like can be performed more suitably (more simply).

In addition, since a three-dimensional cultured cell having a function similar to that of a living body can be obtained by using the base material of the present invention, the three-dimensional cultured cell obtained using the base material described in this specification. Can also be used for regenerative medical applications for various diseases such as diseases in the ophthalmic field such as heart disease, liver disease, and age-related macular degeneration. In the present invention, since cellulose that is safe for living bodies is used as a cell culture substrate, safer three-dimensional cultured cells can be obtained, which is suitable for use in regenerative medicine. That is, the present invention also provides a regenerative medical material containing the three-dimensional cultured cell of the present invention. The present invention also provides a method for treating heart disease, liver disease or ophthalmological disease using the three-dimensional cultured cells of the present invention.

Furthermore, it is possible to transplant both the base material described in the present specification and the three-dimensional cultured cells formed on the base material into the affected part, because a biologically safe material called cellulose is used. . In this way, by transplanting both the base material and the three-dimensional cultured cells on the base material to the affected area, the cells can be easily transplanted to the affected area without being recovered from the base material, and the cells are kept long in the affected area. And the effect of maintaining a high cell survival rate can be expected. That is, the present invention also provides a cell delivery material comprising the cell culture substrate of the present invention and a three-dimensional cultured cell formed on the substrate.

The effect of the present invention will be described using the following examples and comparative examples. However, the technical scope of the present invention is not limited only to the following examples. In the following examples, the operation was performed at room temperature (25 ° C.) unless otherwise specified. Unless otherwise specified, “%” and “part” mean “% by weight” and “part by weight”, respectively.

Example 1
First, cedar wood chips (40 g) were degreased by stirring in a mixture of acetone and water (900 mL: 100 mL (acetone: water)) overnight (15 hours) at room temperature (25 ° C.). It was. Next, this defatted wood chip is delignified by heating in a mixed solution of acetic anhydride and hydrogen peroxide (500 mL: 500 mL (acetic anhydride: hydrogen peroxide)) at 90 ° C. for 2 hours, A holocellulose pulp was obtained. Finally, this holocellulose pulp was immersed in a 5 wt% potassium hydroxide (KOH) aqueous solution at 20 ° C. for 2 hours to obtain an alkali-treated holocellulose pulp suspension.

A high-pressure water jet system (Star Burst, HJP-25005 E, Sugino Machine Co., Ltd.) equipped with a ball-collation chamber was used for 2000 mL of the alkali-treated holocellulose pulp suspension (solid content 0.5 wt%) thus obtained. , Ltd.) was used for high-pressure homogenization (mechanical fibrillation) to obtain a homogenized slurry. This homogenized slurry was extruded from a small nozzle having a diameter of 0.17 mm at a pressure of 245 MPa. The extrusion was repeated 50 times to obtain a high-pressure homogenized cellulose fiber suspension having a concentration of 0.3% by weight, which was designated as coating solution 1 (cellulose fiber / water dispersion).

The coating solution 1 (high-pressure homogenized cellulose fiber concentration: 0.3% by weight) was concentrated to a concentration of 0.8% by weight to prepare a coating solution 1 '. Separately, a glass frame 1 as a support (made by Matsunami Glass Industry Co., Ltd., Sa value: 11.23 nm, thickness: 2 mm) on which a metal frame material with a 7 cm × 7 cm hole is mounted Was prepared (hereinafter referred to as coating plate 1).

The coating solution 1 'prepared above was poured into the holes of the coating plate 1 in an amount of 40 mL / hole. The coating solution 1 ′ was extended all over the hole with a glass rod. After removing the frame material and drying in an oven at 50 ° C. overnight (15 hours), the coating film was recovered from the coating plate 1 to prepare a substrate 1 having a thickness of 30 μm. When the Sa value on the glass plate 1 'surface side of the substrate 1 was measured, it was 125.7 nm (culture surface).

Example 2
In Example 1, instead of the glass plate 1, the same procedure as in Example 1 was used except that an acrylic plate 2 (manufactured by Nippon Test Panel, Sa value: 8.30 nm, thickness: 2 mm) was used as a support. A substrate 2 having a thickness of 30 μm was prepared.

When the Sa value on the surface of the acrylic plate 2 of the base material 2 obtained in this way was measured, it was 86.5 nm (culture surface). Moreover, it was 12.35 nm (culture surface) when the root mean square roughness (RMS value) of the acrylic plate 2 side surface was measured with the atomic force microscope.

Example 3
In Example 1, instead of the glass plate 1, the same procedure as in Example 1 was used except that an acrylic plate 3 (manufactured by Nippon Test Panel, Sa value: 8.17 nm, thickness: 2 mm) was used as a support. A substrate 3 having a thickness of 30 μm was produced.

The Sa value on the surface side of the acrylic plate 3 of the base material 3 obtained in this way was measured and found to be 66.5 nm (culture surface).

Comparative Example 1
In Example 1, it replaced with the glass plate 1, and it carried out similarly to Example 1 except using the acrylic board 4 (The Japan Test Panel company make, Sa value: 30.53 nm, thickness: 2 mm) as a support body. A comparative substrate 4 having a thickness of 30 μm was prepared.

The Sa value on the surface of the acrylic plate 4 of the comparative substrate 4 obtained in this way was measured and found to be 210.7 nm (culture surface).

Comparative Example 2
Wet softwood-dissolved sulfite pulp (dry weight: 20 g) was added distilled water containing 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) (0.16 g) and NaBr (1.0 g) ( (1500 mL) to prepare a cellulose slurry 1. To this cellulose slurry 1, an aqueous sodium hypochlorite (NaClO) solution (4 w / v%) was added at room temperature (25 ° C.) so as to obtain a cellulose concentration of 10 mmol / g while stirring, and 1M sodium hydroxide was added. The mixture was stirred at room temperature (25 ° C.) for 2 hours while maintaining pH 10 to obtain a pulp containing TEMPO-oxidized cellulose.

2000 g of the TEMPO oxidized cellulose suspension (solid content 0.5% by weight) thus obtained was added to a high-pressure water jet system (Star Burst, HJP-25005 E, Sugino Machine Co., equipped with a ball-collation chamber). Ltd.) was used for high-pressure homogenization (mechanical defibration) to obtain a homogenized slurry. This homogenized slurry was extruded from a small nozzle having a diameter of 0.17 mm at a pressure of 245 MPa. The extrusion was repeated 20 times to prepare a coating solution 2 (cellulose fiber / water dispersion) having a concentration of 0.3% by weight.

The coating solution 2 (high-pressure homogenized cellulose fiber concentration: 0.3% by weight) was concentrated to a concentration of 0.8% by weight to prepare a coating solution 2 '.

In Comparative Example 1, a comparative substrate 5 having a thickness of 30 μm was produced in the same manner as in Comparative Example 1, except that the coating liquid 2 ′ prepared above was used instead of the coating liquid 1 ′.

When the Sa value on the acrylic plate 4 (Sa value: 30.53 nm) side of the comparative substrate 5 obtained in this way was measured, it was 249.1 nm (culture surface).

[Performance evaluation]
For the base materials 1 to 3 obtained in Examples 1 to 3 and the comparative base materials 4 to 5 obtained in Comparative Examples 1 and 2, the total light transmittance (%), haze ( %), Static water contact angle (°) and cell attachment behavior.

1. Measurement of total light transmittance (%) The total light transmittance was measured using a haze meter (“HZ-V3” manufactured by Murakami Color Research Laboratory Co., Ltd.) in accordance with JIS standard K7361-1: 1997.

2. Measurement of Haze (%) Haze was measured using a haze meter (“HZ-V3” manufactured by Murakami Color Research Laboratory Co., Ltd.) in accordance with JIS standard K7136: 2000.

3. Measurement of static water contact angle (°) Immediately after 2 μl of water was dropped on the surface (culture surface) on which the Sa value of each substrate was measured using an automatic contact angle meter (manufactured by Kyowa Interface Science: DM-500) The droplet attachment angle was measured (measurement temperature: 25 ° C.). In Table 1 below, it is described as “contact angle”.

4). Evaluation of HepG2 cell adhesion behavior HepG2 (human hepatoma cell) was purchased from DS Pharma Biomedical Co., Ltd. Final concentration of 10 (v / v)% fetal bovine serum (FBS) (manufactured by DS Pharma Biomedical Co., Ltd.), 1/100 volume (volume ratio) of non-essential amino acid for 100 × MEM (DS Pharma Biomedical Co., Ltd.) HepG2 was cultured using an EMEM medium (DS Pharma Biomedical Co., Ltd.) (serum-added EMEM medium) supplemented with a 2 mM glutamine solution (manufactured by DS Pharma Biomedical Co., Ltd.). HepG2 was seeded in a 100 mm cell culture dish (BD Falcon) so as to be 2.0 × 10 ⁴ cells / cm ² and cultured at 37 ° C. under 5 vol% CO ₂ conditions. HepG2 cultured to a confluent state of 70% in a 100 mm cell culture dish was treated with a 0.25% trypsin / 50 mM EDTA solution, and then the serum-added EMEM medium similar to the above was added to stop the trypsin reaction. A suspension cell suspension was obtained. Using 0.4 (w / v)% trypan blue solution (manufactured by Wako Pure Chemical Industries, Ltd.), the number of viable cells in the suspended cell suspension of HepG2 was measured, and 3.13 × 10 ⁴ cells / cm ² Multiwell cell culture plate 24well (BD Falcon, polystyrene), NanoCulture (registered trademark) Plate MS pattern / low adhesion / 24 well (Organogenix), and seeded on each substrate, Culturing was performed under volume% CO ₂ conditions. On the fourth day of culture, the entire medium was removed, and 1 mL of serum-added EMEM medium similar to the above was added to exchange the medium. Culture was performed for a total of 7 days. Each substrate was preliminarily treated with γ-ray sterilization or dry heat sterilization (160 ° C. × 2 hours) and then used for the cell culture.

Also, NanoCulture (registered trademark) Plate MS pattern / low adhesion / 24 wells (Organogenix) have fine irregularities nano-imprinted. For this reason, before seeding the cell suspension, the following deaeration operation was performed to remove bubbles in the irregularities. Specifically, the same serum-added EMEM medium as described above was dispensed at 500 μL per well. This was centrifuged at 300 to 500 × g for 3 minutes and then allowed to stand at room temperature (25 ° C.) for 30 minutes.

On day 7 of culture, HepG2 was treated with 0.25% trypsin / 50 mM EDTA solution, and then the total cell number was measured using a 0.4 w / v% trypan blue solution (Wako Pure Chemical Industries, Ltd.) and a hemocytometer. Went. Further, the culture solution on the seventh day of culture was sampled and stored at -20 ° C.

Fig. 1 shows cell growth conditions (micrographs) on the substrate 1, the comparison substrate 4, the comparison substrate 5, the multiwell cell culture plate and the NanoCulture (registered trademark) Plate on the seventh day of culture.

As an example, the growth state (micrograph) of the cells on the base material 1 on the seventh day of culture is shown in FIG. 1, but the other base materials 2 and 3 had similar results. On the substrates 1 to 3 (Sa value = 12.5 nm (substrate 1), 86.5 nm (substrate 2), 66.5 nm (substrate 3), respectively), spheroids of appropriate size (cell aggregation) In the state where the clumps were attached to the substrate, the cells were uniformly distributed throughout the well, and the expanded cells were hardly observed.

On the other hand, on the comparative base material 4 (Sa value = 210.7 nm), a culture behavior in which spheroids and extended cells coexist was shown. Further, neither spheroid formation nor cell spreading was confirmed on the comparative substrate 5 (Sa value = 249.1 nm).

In addition, in a multiwell cell culture plate 24well (BD Falcon) (multiwell cell culture plate in FIG. 1) generally used for culturing adherent cells, HepG2 cells proliferate in a single layer, and spheroids (cell aggregation) The formation of a lump) was not observed. In addition, in NanoCulture (registered trademark) Plate MS pattern / low adhesion / 24 well (Organogenix) (NanoCulture Plate in FIG. 1), spheroids (cell aggregates) were formed, but most of the cell aggregates were base materials It floated in the medium without adhering to the cells, and the suspended cell aggregates formed a larger mass. Furthermore, cell aggregates attached to the base material were also associated to form a large spheroid.

5). Evaluation of MCF-7 cell adhesion behavior MCF-7 (human breast cancer cells) was purchased from DS Pharma Biomedical Co., Ltd. Final concentration of 10 (v / v)% fetal bovine serum (FBS) (manufactured by DS Pharma Biomedical Co., Ltd.), 1/100 volume (volume ratio) of non-essential amino acid for 100 × MEM (DS Pharma Biomedical Co., Ltd.) MCF-7 was cultured using EMEM medium (DS Pharma Biomedical) (serum-added EMEM medium) supplemented with 2 mM glutamine solution (manufactured by DS Pharma Biomedical). . MCF-7 was seeded in a 100 mm cell culture dish (BD Falcon) at 2.0 × 10 ⁴ cells / cm ² and cultured at 37 ° C. under 5 vol% CO ₂ conditions. MCF-7 cultured to a confluent state of 70% in a 100 mm cell culture dish was treated with a 0.25% trypsin / 50 mM EDTA solution, and then the serum-added EMEM medium similar to the above was added to stop the trypsin reaction. A suspension of MCF-7 suspension was obtained. Using a 0.4% (w / v)% trypan blue solution (manufactured by Wako Pure Chemical Industries, Ltd.), the number of viable cells in the suspended cell suspension of MCF-7 was measured, and 3.13 × 10 ⁴ cells / cm ² in a multi-well cell culture plate 24well (BD Falcon, polystyrene), NanoCulture (registered trademark) Plate MS pattern / low adhesion / 24 well (ORGANOGENIX), and each substrate, 37 ° C. And cultured under 5 vol% CO ₂ conditions. On the fourth day of culture, the entire medium was removed, and 1 mL of serum-added EMEM medium similar to the above was added to exchange the medium. Culture was performed for a total of 7 days. In addition, the following base material was used for the cell culture after γ-ray sterilization treatment or dry heat sterilization treatment (160 ° C. × 2 hours) in advance.

Also, NanoCulture (registered trademark) Plate MS pattern / low adhesion / 24 wells (Organogenix) have fine irregularities nano-imprinted. For this reason, before seeding the cell suspension, the following deaeration operation was performed to remove bubbles in the irregularities. Specifically, the same serum-added EMEM medium as described above was dispensed at 500 μL per well. This was centrifuged at 300 to 500 × g for 3 minutes and then allowed to stand at room temperature (25 ° C.) for 30 minutes.

FIG. 2 shows cell growth conditions (micrographs) on the substrate 1, the comparison substrate 4, the comparison substrate 5, the multiwell cell culture plate and the NanoCulture (registered trademark) Plate on the seventh day of culture.

As an example, the cell growth state (micrograph) on the base material 1 on the seventh day of culture is shown in FIG. 2, but the other base materials 2 and 3 had similar results. On the substrates 1 to 3 (Sa value = 12.5 nm (substrate 1), 86.5 nm (substrate 2), 66.5 nm (substrate 3), respectively), spheroids of appropriate size (cell aggregation) In the state where the clumps were attached to the substrate, the cells were uniformly distributed throughout the well, and the expanded cells were hardly observed. The MCF-7 cells used here are less likely to be three-dimensional than the HepG2 cells used in “4. Evaluation of HepG2 cell attachment behavior” above, but these cells are also difficult to make three-dimensional. It is considered that by culturing on the base material of the present invention, spheroids (cell aggregates) having an appropriate size can be formed while attached to the base material.

On the other hand, almost no spheroid formation was confirmed on the comparative substrate 4 (Sa value = 210.7 nm), and most of the cells were stretched cells. Further, neither spheroid formation nor cell spreading was confirmed on the comparative substrate 5 (Sa value = 249.1 nm).

In addition, in the multiwell cell culture plate 24well (BD Falcon) (multiwell cell culture plate in FIG. 2) generally used for culturing adherent cells, MCF-7 cells proliferate in a single layer, and spheroids ( The formation of cell aggregates was not observed. In addition, in NanoCulture (registered trademark) Plate MS pattern / low adhesion / 24 wells (Organogenix) (NanoCulture Plate in FIG. 2), spheroids (cell aggregates) were formed, but spread and adhered to the substrate. Many cells were also observed.

These results are summarized in Table 1 below.

From the results of Table 1 above, it is shown that spheroids can be selectively formed on the substrate by culturing the cells on the substrates 1 to 3 of Examples 1 to 3. On the other hand, when cells are cultured on the comparative substrates 4 to 5 of Comparative Examples 1 and 2 in which the Sa value of the culture surface is outside the numerical range of the present invention, spheroids cannot be selectively formed.

6). Albumin Quantification Regarding the substrate 1 obtained in Example 1 above, and the commercially available multiwell cell culture plate 24well (BD Falcon) and NanoCulture (registered trademark) Plate MS pattern / low adhesion / 24 well (ORGANOGENIX) According to the method, the amount of albumin produced was measured.

Specifically, the culture was performed for 7 days in the same manner as in “4. Evaluation of HepG2 cell adhesion behavior”. Culture on Multiwell Cell Culture Plate 24well (BD Falcon) on Day 7 of Culture, NanoCulture (R) Plate MS Pattern / Low Adhesion / 24 Well (Organogenix), and Substrate 1 (Sa Value = 15.7 nm) Using the culture broth, the amount of albumin produced (μg / 10 ⁶ cells) was measured. In addition, for the quantification of albumin, Rat Albumin ELISA Quantitation Set (Bethyl Laboratories) was used, and quantification experiment of albumin was performed by double measurement according to the attached protocol.

The results are shown in FIG. From FIG. 3, the amount of albumin produced in the culture medium cultured on the substrate 1 (Sa value = 125.7 nm) was significantly higher than the culture medium cultured on the multiwell cell culture plate and NanoCulture (registered trademark) Plate. It can be seen that was confirmed. The above results and 4. From the evaluation results of the HepG2 cell attachment behavior, medium components and oxygen can be efficiently supplied to the cells and high cell functions can be maintained by forming a cell aggregate of appropriate size on the substrate of the present invention. It is considered. From these results, it is expected that cells can express high functions by culturing the cells with the substrate of the present invention.

7). Examination of the relationship between the content of cellulose fibers having a diameter of 1 μm or more and transparency (haze) In this experiment, the relationship between the content (%) of cellulose fibers having a diameter of 1 μm or more and transparency (haze) was examined. .

First, the contents of cellulose fibers having a diameter of 1 μm or more were measured for the substrates A to C produced as described below by the method described above.

(Preparation of substrate A)
Conifer dissolved sulfite pulp / water suspension (solid content 0.5 wt%) using a high pressure water jet system (Star Burst, HJP-25005 E, Sugino Machine Co., Ltd.) equipped with a ball-collation chamber. Then, high-pressure homogenization (mechanical defibration) was performed to obtain a homogenized slurry. This homogenized slurry was extruded from a small nozzle having a diameter of 0.17 mm at a pressure of 245 MPa. The above extrusion (defibration treatment) was repeated 50 times to obtain a nanofiber suspension A. The nanofiber aqueous suspension A was suction filtered and dried to prepare a substrate A having a thickness of 40 μm.

The base material A thus obtained was measured for haze, which was 38.3%. Moreover, about this base material A, when the content rate of the cellulose fiber whose diameter is 1 micrometer or more was measured, it was 0.28%.

(Preparation of base material B)
In the same manner as in Example 1, an alkali-treated holocellulose pulp suspension was produced. The alkali-treated holocellulose was separated from the suspension thus obtained, suspended in water to a concentration of 0.5% by weight, and the alkali-treated holocellulose / water suspension (solid content 0.5). % By weight) was prepared.

This alkali-treated holocellulose / water suspension (solid content 0.5% by weight) was subjected to a high-pressure water jet system (Star Burst, HJP-25005 E, Sugino Machine Co., Ltd.) equipped with a ball-collation chamber. Using, high-pressure homogenate (mechanical fibrillation) to obtain a homogenized slurry. This homogenized slurry was extruded from a small nozzle having a diameter of 0.17 mm at a pressure of 245 MPa. The above extrusion (defibration treatment) was repeated 50 times to obtain a nanofiber suspension B. The nanofiber aqueous suspension B was suction filtered and dried to produce a substrate B having a thickness of 40 μm.

For the base material B thus obtained, the haze was measured and found to be 5.5%. Moreover, about this base material B, when the content rate of the cellulose fiber whose diameter is 1 micrometer or more was measured, it was 0.05%.

(Preparation of substrate C)
Cellulose powder / water suspension (solid content 0.5 wt%) was subjected to high pressure using a high pressure water jet system (Star Burst, HJP-25005 E, Sugino Machine Co., Ltd.) equipped with a ball-collation chamber. Homogenated (mechanical fibrillation) to obtain a homogenized slurry. This homogenized slurry was extruded from a small nozzle having a diameter of 0.17 mm at a pressure of 245 MPa. The extrusion (defibration treatment) was repeated 50 times to obtain a nanofiber suspension C. The nanofiber aqueous suspension C was suction filtered and dried to prepare a substrate C having a thickness of 40 μm. As the cellulose powder, KC Flock (registered trademark) W100-GK (Nippon Paper Group) was used.

The base material C thus obtained was measured for haze and found to be 86.6%. Moreover, about this base material C, when the content rate of the cellulose fiber whose diameter is 1 micrometer or more was measured, it was 10.19%.

The results are summarized in Table 2 below. From Table 2 below, a positive correlation is recognized between the content of cellulose fibers having a diameter of 1 μm or more and haze. From the above results, it is considered that the transparency of the substrate can be significantly improved by reducing the ratio of cellulose fibers having a diameter of 1 μm or more to the substrate.

8). Examination of the relationship between the content of cellulose fibers having a diameter of 1 μm or more and the ease of visual observation of cell culture In this experiment, the relationship between the content (%) of cellulose fibers having a diameter of 1 μm or more and the ease of visual observation of cell cultures investigated.

First, substrates A to C were prepared in the same manner as in “7. Examination of relationship between content of cellulose fiber having diameter of 1 μm or more and transparency (haze)”.

Next, HepG2 cells were cultured on the substrates A to C for 7 days in the same manner as in “4. Evaluation of HepG2 cell adhesion behavior”. The HepG2 cell culture after culturing for a predetermined period was observed under a microscope (magnification: 40 times). As a result, since the substrates A and B are excellent in transparency, the growth state of the HepG2 cells can be easily observed visually, but the substrate C is inferior in transparency and the observation image is unclear, It was difficult to observe the growth state of HepG2 cells. From the above results, it is considered that the ease of visual observation of cells can be significantly improved by reducing the ratio of cellulose fibers having a diameter of 1 μm or more to the substrate.

9. Measurement of albumin adsorption amount With respect to the substrate 1 produced in the same manner as in Example 1, the albumin adsorption amount was measured according to the method described below.

Bovine serum albumin (Sigma; A8022-10G) was dissolved in Dulbecco's PBS (-) (Wako; 041-20211) to an albumin concentration of 20 μg / mL to obtain an albumin solution. A film obtained by cutting the substrate 1 into a circle having a diameter of 14 mm was set on a pedestal on a petri dish, and 300 μL of an albumin solution was mounted on the film.

Put another film of the same type (circular with a diameter of 14 mm) on the albumin solution, sandwich the albumin solution between the two films, set the area where the film and the albumin solution contact each other, and set the unit Obtained. This was left still in a 5 volume% CO ₂ incubator at 37 ° C. containing a vat containing water and humidified. After 7 hours, the unit was taken out and the albumin solution was recovered. The collected solution was stored at −20 ° C. until measurement.

The albumin concentration in the collected albumin solution was measured. The concentration was measured using Albamin, Bovine, ELISA Quantification kit (Bethyl Laboratories), and the measurement protocol was in accordance with the attached manual. From the initial concentration (20 [mu] g / mL) area and the difference in the film between the albumin concentration after 7 hours (one per side 1.54cm ² × 3.08cm ² with two sheets), albumin adsorption amount per unit area ( ng / cm ²⁾ was calculated, was 1037ng / cm ^2.

10. Measurement of adsorption amount of proteoglycan The adsorption amount of proteoglycan was measured for the substrate 1 produced in the same manner as in Example 1 according to the method described below.

Heparan sulfate proteoglycan (Sigma; H4777) was dissolved in Dulbecco's PBS (-) (Wako; 041-20211) so that the proteoglycan concentration was 5 μg / mL to obtain a proteoglycan solution. A film obtained by cutting the substrate 1 into a circle having a diameter of 14 mm was set on a pedestal on a petri dish, and 300 μL of a proteoglycan solution was mounted on the film.

Place the same type of film (circular with a diameter of 14 mm) on the proteoglycan solution, sandwich the proteoglycan solution between the two films, set the area where the film and the proteoglycan solution are in contact with each other, and set the unit. Obtained. This was left still in a 5 volume% CO ₂ incubator at 37 ° C. containing a vat containing water and humidified. After 20 hours, the unit was taken out and the proteoglycan solution was recovered. The collected solution was stored at −20 ° C. until measurement.

The proteoglycan concentration in the collected proteoglycan solution was measured. The concentration was measured using Glycosaminoglycan Sulfurated Alcian Blue Binding Assay (Euro Diagnostics), and the measurement protocol was based on the attached manual. From the difference between the initial concentration (5 μg / mL) and the proteoglycan concentration after 20 hours and the area of the film (1.54 cm ² × 2 sheets per sheet, 3.08 cm ² ), the amount of proteoglycan adsorbed per unit area ( ng / cm ²⁾ was calculated and found to be 162.6ng / cm ^2.

11. Evaluation of rat primary hepatocyte adhesion behavior A Wistar rat, male, 9 weeks old, 200 g body weight of Special Viral Pathogen Free was purchased from SLC Japan. Rat primary hepatocytes were obtained by referring to the method described in Chapter 10 of the Cultured Cell Experiment Handbook (Yodosha), “Hepatocytes”. Specifically, Wistar rats were laparotomized under isoflurane anesthesia, a catheter was inserted into the portal vein, and a preperfusion solution having the composition shown in Table 3 was injected. The chest cavity was then opened and the inferior vena cava entering the right atrium was incised to release blood. After confirming that blood removal from the liver was sufficiently performed, the perfusion was stopped, and the perfusion solution was replaced with a collagenase solution having the composition shown in Table 3 for perfusion. After confirming that the intercellular tissue was digested by collagenase, the perfusion was stopped. After the liver was cut off and transferred to a glass petri dish, cold EMEM High Glucose medium (Wako) was added to disperse the cells by pipetting. Next, undigested tissue was removed with a 150 mm filter. The cell suspension was centrifuged at 50G for 1 minute several times to remove non-parenchymal cells. The survival rate of the obtained hepatocytes was measured by the trypan blue exclusion method, and hepatocytes with a survival rate of 85% or more were used as rat primary hepatocytes in the culture test.

The rat primary hepatocytes obtained by the above-described method were suspended in a serum medium having the composition described below, and obtained in Example 1 above, which was sterilized with gamma rays so as to be 1.33 × 10 ⁴ cells / cm ² . were seeded on the substrate 1, 37 ° C., the cells were cultured under 5% CO _2. The medium was exchanged 4 hours after seeding, after removing the entire medium on the first day, the third day, and the fifth day of culture, and then adding 0.4 mL of serum medium. On day 5 of culture, spheroid formation and the presence or absence of extended cells were confirmed. FIG. 5 shows the growth state (micrograph) of the cells on the substrate 1 on the fifth day of culture. In the substrate 1, almost no stretched cells were observed, and it was confirmed that spheroids (cell aggregates) having a diameter of 200 μm or less were uniformly distributed throughout the well in a state of adhering to the substrate.

(Composition of serum medium)
William's E medium (Wako Pure Chemical), 10% (w / v) FBS (Wako Pure Chemical), 8.6 nM insulin, 255 nM dexamethasone, 50 ng / mL EGF, 5KIU / mL aprotinin, antibiotics (penicillin (100 units / mL) / streptomycin (100 μg / mL) / amphotericin B (0.25 μg / mL)).

This application is based on Japanese Patent Application No. 2016-203818 filed on October 17, 2016, the disclosure of which is referenced and incorporated as a whole.

1 ... Substrate for cell culture (film),
20 ... support member,
100, 101, 102 ... Cell culture container.

Claims (9)

A cell culture substrate containing cellulose fibers and having an arithmetic average height (Sa) of the culture surface of less than 210 nm. The base material according to claim 1, wherein the haze is 40% or less. The substrate according to claim 1 or 2, wherein the content of cellulose fibers having a diameter of 1 µm or more is less than 10%. The cell culture substrate according to any one of claims 1 to 3, which comprises applying a coating solution containing cellulose fibers onto a support having an arithmetic average height (Sa) of the culture surface of less than 30 nm. Manufacturing method. A culture container having the cell culture substrate according to any one of claims 1 to 3. A method for culturing cells, comprising culturing cells on the culture surface of the cell culture substrate according to any one of claims 1 to 3. The method according to claim 6, wherein the culture is a three-dimensional culture. A three-dimensional cultured cell formed on the cell culture substrate according to any one of claims 1 to 3. A method for testing a drug in vitro, comprising using the three-dimensional cultured cell according to claim 8.

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