JP7841541B2 - Cell culture vessel, cell culture method, and method for manufacturing a cell culture vessel - Google Patents

Description

This invention relates to a cell culture vessel. This invention also relates to a cell culture method using a cell culture vessel.

Cells cultured in three dimensions can sometimes exhibit functions closer to those in vivo compared to those cultured in two dimensions. To culture cells in three dimensions, for example, a scaffold is used. Patent document 1 (Japanese Patent Publication No. 2018-113945) discloses a method for forming spheroids by contacting gelatin with cells. Patent document 2 (International Publication No. 2018/142633) discloses the use of a gel composition containing an amphiphilic block polymer as a scaffold for three-dimensional cell culture.

Japanese Patent Publication No. 2018-113945 International Publication No. 2018/142633

When cells are cultured in three dimensions, it is preferable to form a uniform cell aggregate depending on the type and application of the cells. When the cell aggregate is uniform, cell death inside the cell aggregate is easily suppressed, and differences in cell properties and types between the inner and outer cells are less likely to occur. The present invention aims to obtain a cell aggregate with small size variations.

One embodiment of the present invention is
A cell culture vessel having multiple recesses,
The aforementioned recess decreases in diameter from the opening towards the bottom.
The present invention relates to a cell culture vessel in which the recess is covered with a gel composition containing an amphiphilic block polymer having hydrophilic block chains and hydrophobic block chains.

One embodiment of the present invention is
A cell culture method using a cell culture vessel,
A step of preparing a cell culture vessel having a recess, wherein a gel composition is applied to the recess,
The present invention relates to a method comprising the step of wetting the gel composition.

One embodiment of the present invention is
A method for manufacturing a cell culture vessel,
The process includes the step of applying a gel composition to the recess of the cell culture vessel having a recess,
The present invention relates to a method for producing the gel composition, which comprises an amphiphilic block polymer having hydrophilic block chains and hydrophobic block chains.

According to the present invention, cell aggregates with small size variations can be stably obtained.

This diagram illustrates a three-dimensional network formed by amphiphilic block polymers. This figure shows an example of a cell culture method according to the present invention. These are photographs showing cell aggregates formed in wells without a gel composition coating (left figure) and with a gel composition coating (right figure) in Experiment 2.

[Cell culture container]
A cell culture vessel according to one embodiment of the present invention is
A cell culture vessel having multiple recesses,
The aforementioned recess decreases in diameter from the opening towards the bottom.
The recess is covered with a gel composition containing an amphiphilic block polymer having hydrophilic block chains and hydrophobic block chains.

The cell culture vessel has a recess that narrows in diameter from the opening to the bottom, which encourages cells to gather at the bottom of the recess and facilitates the formation of uniformly sized cell aggregates (spheroids). A gel composition containing an amphiphilic blocking polymer covering the recess assists cell aggregation, further promoting cell aggregate formation. Uniformly formed cell aggregates have stable quality and are more likely to perform their intended function.

(Cell culture vessel with multiple recesses)
The cell culture vessel according to the present invention has a plurality of recesses. The recesses narrow in diameter from the opening towards the bottom. Cells gather in one place on the bottom surface of the recesses due to gravity. One cell aggregate can be formed within one recess. The recesses may be narrowed in diameter from the opening towards the bottom, either entirely or in part. The recesses may have portions where the diameter does not change from the opening towards the bottom. Preferably, the plurality of recesses have a uniform size and shape.

Examples of cell culture vessels according to the present invention include multi-well plates, dishes, and flasks, with multi-well plates or dishes having 6 to 384 wells being preferred.

The cell culture vessel according to the present invention can be molded from a resin material. This resin material allows for the container to be disposable and can be easily molded into various shapes. Examples of resin materials include polyolefin resins such as polypropylene resin, polyethylene resin, and ethylene-propylene copolymer, or cyclic polyolefin resins; polystyrene resins such as polystyrene and acrylonitrile-butadiene-styrene resin; methacrylic resins such as polycarbonate resin, polyethylene terephthalate resin, and polymethyl methacrylate resin; vinyl chloride resin, polybutylene terephthalate resin, polyarylate resin, polysulfone resin, polyethersulfone resin, polyetheretherketone resin, polyetherimide resin, and fluorine-based resins such as polytetrafluoroethylene; acrylic resins such as polymethylpentene resin and polyacrylonitrile; and cellulose-based resins such as propionate resin. Among these, from the viewpoint of moldability and sterilization properties required for culture vessels, it is preferable that the cell culture vessel contains polystyrene resin.

It is preferable that the recesses of the cell culture vessel are non-adherent to cells. Methods for making the recesses non-adherent to cells include hydrophilic treatment of the surface of the recesses with phospholipid polymers (such as 2-methacryloyloxyethyl phosphorylcholine), polyhydroxyethyl methacrylate, fluorine-containing compounds, polyethylene glycol, etc., and low protein adsorption treatment. The recesses may also be formed from a non-adherent resin, for example, the cell culture vessel may be molded from a silicone resin. In one example of a cell culture vessel, at least the recesses are made of polystyrene resin and the surface is hydrophilic. In the cell culture vessel according to the present invention, a gel composition is further laminated on the surface of the hydrophilic treated recesses, but the gel composition separates from the recess surface during cell culture. If the exposed recess surface is non-adherent to cells, cells tend to aggregate inside the recesses to form cell aggregates, and the cell aggregates can be easily removed.

(1) An example of a recess is an irregularity provided on the bottom surface (culture surface) of the portion that contains the culture medium. In this case, the culture surface is usually flat. The cell culture vessel may be a multi-well plate or a dish. A preferred example of the cell culture vessel according to the present invention is a plate in which the flat bottom surface of each well (portion that contains the culture medium) of a multi-well plate such as a 6-well, 12-well, or 24-well plate is provided with irregularities. A preferred example of the cell culture vessel according to the present invention is a dish in which the culture surface of a cell culture dish such as a 35 mm dish, a 60 mm dish, or a 100 mm dish is provided with irregularities. The irregularity shape is preferably composed of a continuous curved surface.

For example, more than 10 depressions are formed on the culture surface. For example, a circular culture surface with a diameter of 85 mm can form approximately 14,200 depressions (about 250 depressions/ ^cm² ), a circular culture surface with a diameter of 35 mm can form approximately 2,700 depressions, and a circular culture surface with a diameter of 100 mm can form approximately 17,000 depressions. Preferably, 10 or more depressions/ ^cm² , 15 or more depressions/ ^cm² , or 20 or more depressions can be formed per unit area of the culture surface, and 10,000 or less depressions/ ^cm² , 5,000 or less depressions/ ^{cm² ,} or 1,000 or less ^depressions can be formed.

The recesses are formed, for example, by irradiating the culture surface with laser light. Specifically, assuming the x-y axis is the plane direction of the culture surface, first, the irradiation unit of the laser irradiation device is scanned in the positive x-axis direction, and laser light is irradiated at regular intervals (e.g., 800 μm) to form multiple recesses aligned in the x-axis direction. Subsequently, the irradiation unit is scanned in the y-axis direction for a certain distance (e.g., 400 μm), and then the irradiation unit is scanned in the negative x-axis direction, and laser light is irradiated at regular intervals (e.g., 800 μm) to form multiple recesses aligned in the x-axis direction. Similarly, the irradiation unit is scanned in the y-axis direction for a certain distance (e.g., 400 μm). By repeating this process, multiple regularly arranged recesses can be formed on the culture surface. To make the size of the recesses uniform, it is preferable to scan the irradiation unit of the laser irradiation device without changing the laser output and irradiation speed.

When forming a recess with a laser, a _CO2 laser may be used as the laser light source, and the laser light may be pulsed at an output of 10 W and an irradiation speed of 6100 mm/min. The shape of the irradiation spot is circular, and its diameter is, for example, 20 μm to 1500 μm, and may be about 400 μm.

When a laser beam is shone on the culture surface, the synthetic resin material constituting the culture surface dissolves and vaporizes, making it possible to form depressions with smooth surfaces. Around the opening of the depressions, the dissolved synthetic resin material may rise up, forming a ridge. Two adjacent depressions, and the peripheral walls of wells adjacent to depressions, are formed by one or more ridges, and it is preferable that no flat surfaces remain between adjacent depressions, and between peripheral walls adjacent to depressions and depressions. In other words, it is preferable that the culture surface between adjacent depressions, and between peripheral walls adjacent to depressions and depressions, is composed of a continuous curved surface. It is preferable that the entire culture surface has irregularities, but parts not used for culture may have flat surfaces.

The depth of the recess may be adjusted as appropriate depending on the number of cells and the size of the cell aggregate, and is usually between 10 μm and 1500 μm, may be 100 μm or more, may be 1000 μm or less, 900 μm or less, or 500 μm or less, and may be around 150 μm to 200 μm.

The shape of the opening may be circular or elliptical, or it may be polygonal (triangle, quadrilateral, pentagon, hexagon, octagon, etc.). The major axis of the opening surface may be, for example, 10 μm or more and 2000 μm or less, 100 μm or more or 200 μm or more, 1500 μm or less or 1000 μm or less, or about 400 μm to 500 μm.

Examples of commercially available cell culture vessels with uneven surfaces include "EZSPHERE" (manufactured by AGC Technoglass Co., Ltd.) and Elplasia (manufactured by Corning).

(2) An example of a cell culture vessel having multiple recesses is a multiwell plate (microwell plate) in which the vertical cross-sectional shape of the bottom of the wells is conical (V-shaped), frustoconical, or hemispherical (U-shaped). The plate may be a 96-well, 384-well, or 1536-well plate.

Examples of commercially available cell culture vessels with multiple recesses include "PrimeSurface" (manufactured by Sumitomo Bakelite Co., Ltd.) and "Nunclon Sphere" (manufactured by Thermo Fisher Scientific).

(Gel composition containing amphiphilic block polymer)
The gel composition according to the present invention is a composition whose main component is an amphiphilic block polymer having hydrophilic block chains and hydrophobic block chains. The gel composition may be a xerogel substantially free of dispersion medium, or it may be wetted with water as a dispersion medium. An organogel is obtained by mixing the amphiphilic block polymer with an organic solvent, and a xerogel is formed by removing the organic solvent from the organogel. The gel composition is wetted by mixing the xerogel with water, forming, for example, a hydrosol or hydrogel. The gel composition according to the present invention can be maintained as a xerogel free of solvent (dispersion medium) and can be stored at room temperature. The gel composition according to the present invention is useful as a scaffold material for three-dimensional cell culture.

When an amphiphilic block polymer is mixed with an organic solvent, the polymer self-assembles, forming a rod structure with hydrophobic block chains facing inward. Even when the organic solvent is removed, the rod structure is thought to be maintained as a xerogel. When the xerogel is moistened by the addition of water, the gel composition is thought to swell and form a fibrous three-dimensional network (Figure 1). Cells physically aggregate within this three-dimensional network. The water-moistened gel composition does not need to have cell-adherent properties; preferably, it has non-cell-adherent properties, allowing cells to repel each other from the three-dimensional network formed by the polymer, thus assisting in cell aggregation.

Since the gel composition according to the present invention is not covalently bonded to the recesses, it can be separated from the surface of the recesses when wetted. The amphiphilic block polymer and cell aggregates can be easily recovered from the cell culture vessel. The cell aggregates may be recovered by further adding water to the amphiphilic block polymer and cell aggregates to break down the three-dimensional network of the amphiphilic block polymer.

Because the gel composition according to the present invention is based on a synthetic polymer, it is less likely to contain unknown components and less likely to exhibit lot-to-lot variations compared to gel compositions based on natural (e.g., animal) derived proteins. Therefore, the gel composition according to the present invention has excellent quality stability. Because the gel composition according to the present invention is based on a biodegradable synthetic polymer, it can be applied to living organisms such as humans and can be used in the field of tissue culture for regenerative medicine. Furthermore, the gel composition according to the present invention can also be used as a cell protective material or cell preservation substrate when transplanting cultured cells.

Examples of monomer units for hydrophilic block chains include alkylene oxides and sarcosine. Examples of monomer units for hydrophobic block chains include hydroxy acids such as glycolic acid, lactic acid, and hydroxyisobutyric acid, and hydrophobic amino acids or their amino acid derivatives such as glycine, alanine, valine, leucine, isoleucine, proline, methionine, tyrosine, tryptophan, methyl glutamate, benzyl glutamate, methyl aspartate, ethyl aspartate, and benzyl aspartate. In particular, amphiphilic block polymers in which the hydrophilic block chain contains sarcosine units and the hydrophobic block chain contains lactic acid units tend to form gels suitable for cell culture.

(Hydrophilic block chain)
Hydrophilic block chains preferably contain 20 or more sarcosine units (N-methylglycine units). Sarcosine has high water solubility. Furthermore, polysarcosine has an N-substituted amide, which allows for cis-trans isomerization, and has high flexibility due to minimal steric hindrance around the α-carbon. Therefore, by using polysarcosine chains as constituent units, hydrophilic block chains possessing both high hydrophilicity and flexibility are formed.

If a hydrophilic block chain has 20 or more sarcosine units, adjacent hydrophilic blocks of block polymers tend to aggregate, making it easier to form a gel that incorporates a hydrophilic dispersion medium such as water or alcohol. There is no particular upper limit to the number of sarcosine units in a hydrophilic block chain. From the viewpoint of stabilizing the gel structure by agglomerating hydrophobic blocks of amphiphilic polymers of adjacent block polymers, the number of sarcosine units in a hydrophilic block chain is preferably 300 or less. The number of sarcosine units is more preferably 25 to 200, and even more preferably 30 to 150.

The hydrophilic block chain may have all sarcosine units continuous, or the sarcosine units may be discontinuous as long as the properties of polysarcosine described above are not impaired. If the hydrophilic block chain has monomer units other than sarcosine, the monomer units other than sarcosine are not particularly limited, but examples include hydrophilic amino acids or amino acid derivatives. The amino acids include α-amino acids, β-amino acids, and γ-amino acids, and are preferably α-amino acids. Examples of hydrophilic α-amino acids include serine, threonine, lysine, aspartic acid, and glutamic acid. The hydrophilic block may also have sugar chains or polyethers. It is preferable that the hydrophilic block has a hydrophilic group such as a hydroxyl group at its terminal (the terminal opposite the linker portion to the hydrophobic block).

(Hydrophobic block chain)
The hydrophobic block chain preferably contains 10 or more lactic acid units. Polylactic acid has excellent biocompatibility and stability. Furthermore, because polylactic acid has excellent biodegradability, it is rapidly metabolized and has low accumulation in the body. For this reason, amphiphilic polymers with polylactic acid as a constituent block are useful for applications in living organisms, especially the human body. In addition, because polylactic acid is crystalline, even if the hydrophobic block chain is short, the hydrophobic block chain aggregates in a solvent such as alcohol, and gel formation is easily achieved.

There is no particular upper limit to the number of lactic acid units in the hydrophobic block chain, but from the viewpoint of stabilizing the structure, it is preferable to have 1000 or fewer units. The number of lactic acid units in the hydrophobic block is preferably 10 to 1000, more preferably 15 to 500, and even more preferably 20 to 100.

The lactic acid units constituting the hydrophobic block chain may be either L-lactic acid or D-lactic acid. Furthermore, a mixture of L-lactic acid and D-lactic acid may be present. The hydrophobic block chain may consist entirely of continuous lactic acid units, or the lactic acid units may be discontinuous. The monomer units other than lactic acid included in the hydrophobic block chain are not particularly limited, but examples include hydroxy acids such as glycolic acid and hydroxyisobutyric acid, and hydrophobic amino acids or their amino acid derivatives such as glycine, alanine, valine, leucine, isoleucine, proline, methionine, tyrosine, tryptophan, methyl glutamate, benzyl glutamate, methyl aspartate, ethyl aspartate, and benzyl aspartate.

(Structure and synthesis methods of amphiphilic block polymers)
Amphiphilic polymers are formed by linking hydrophilic block chains and hydrophobic block chains. The hydrophilic and hydrophobic block chains may be linked via a linker. Preferably, the linker has a functional group (e.g., hydroxyl group, amino group, etc.) that can bond to lactic acid monomers (e.g., lactic acid or lactide) or polylactic acid chains, which are constituent units of the hydrophobic block chain, and a functional group (e.g., amino group) that can bond to sarcosine monomers (e.g., sarcosine or N-carboxysarcosine anhydride) or polysarcosine, which are constituent units of the hydrophilic block chain. By appropriately selecting the linker, the branching structure of the hydrophilic and hydrophobic block chains can be controlled.

The method for synthesizing amphiphilic block polymers is not particularly limited, and known peptide synthesis methods, polyester synthesis methods, depsipeptide synthesis methods, etc., can be used. For details, amphiphilic block polymers can be synthesized by referring to WO2009/148121, etc.

To adjust the hardness, stability, and degradability (solubility) of the gel, it is preferable to adjust the chain length of the hydrophobic block chain (number of lactic acid units) and the ratio of the chain lengths of the hydrophobic block chain to the hydrophilic block chain (ratio of the number of lactic acid units to the number of sarcosine units). To facilitate the control of the chain length of the hydrophobic block chain, it is preferable to first synthesize a hydrophobic block chain (e.g., polylactic acid) with a linker introduced at one end, and then introduce a hydrophilic block chain (e.g., polysarcosine) during the synthesis of the amphiphilic block polymer. The chain lengths of the hydrophobic and hydrophilic block chains can be adjusted by adjusting conditions such as the initial-to-monomer ratio, reaction time, and temperature in the polymerization reaction. The chain lengths of the hydrophilic and hydrophobic block chains (molecular weight of the amphiphilic block polymer) can be confirmed, for example, by 1H-NMR. From the viewpoint of improving the biodegradability of the amphiphilic polymer, the weight-average molecular weight is preferably 10,000 or less, and more preferably 9,000 or less. The amphiphilic polymer used in the present invention may have chemical crosslinks formed between its molecules for purposes such as promoting gel formation and improving gel stability.

(Preparation of gel composition)
For forming organogels, a solvent that readily dissolves the hydrophilic block chains of an amphiphilic polymer and poorly dissolves the hydrophobic block chains is preferred. For example, for an amphiphilic block polymer containing polylactic acid as the hydrophobic block chain and polysarcosine as the hydrophilic block chain, an organic solvent that dissolves polysarcosine but not polylactic acid is preferred. By using such an organic solvent, the hydrophobic block portions of the amphiphilic polymer aggregate under the mixture of the amphiphilic polymer and the organic solvent, making it easier to form a physically crosslinked matrix. Furthermore, if an organogel is formed using such an organic solvent, the xerogel after removal of the organic solvent also tends to adopt a structure in which the hydrophobic block portions aggregate. Therefore, when water is brought into contact with the xerogel, water easily penetrates the hydrophilic block chain portions, and it is thought that a hydrosol or hydrogel having a polymer matrix structure similar to that of an organogel is formed.

Alcohols having 1 to 6 carbon atoms are preferred as organic solvents used for organogel formation. Among these, alcohols having 1 to 4 carbon atoms are preferred because they offer high solubility for hydrophilic block chains and facilitate the formation of xerogels by removing the organic solvent. Specific examples of preferred organic solvents include methanol, ethanol, propanol, 2-propanol, and butanol.

Two or more organic solvents may be used in mixture form. The solubility of hydrophobic and hydrophilic block chains may be adjusted by mixing two or more organic solvents. After dissolving the amphiphilic polymer with a highly soluble organic solvent, adding an organic solvent with low solubility for hydrophobic block chains can promote physical crosslinking by aggregation of hydrophobic blocks, thereby forming a gel matrix. When two or more organic solvents are used, it is preferable that at least one of them is the alcohol described above. Two or more alcohols may be used. When the organic solvent is a mixed solvent of two or more organic solvents, it is preferable that 50% by weight or more of the total amount of organic solvent is the alcohol described above. The amount of alcohol relative to the total amount of organic solvent is more preferably 60% by weight or more, and even more preferably 70% by weight or more.

The ratio of the amphiphilic polymer to the organic solvent is not particularly limited and should be set within a range that allows the amphiphilic polymer to dissolve or swell, depending on the molecular weight of the amphiphilic polymer and the type of organic solvent. From the viewpoint of appropriately maintaining the distance between adjacent amphiphilic polymers and suppressing gel formation, the amount of organic solvent is preferably 100 to 1500 parts by weight, and more preferably 200 to 1000 parts by weight, per 100 parts by weight of amphiphilic polymer. The content of amphiphilic block polymer in the organogel composition is preferably 10% by weight or more.

In organogel formation, it is preferable to prepare a fluid, viscous liquid by dissolving or swelling the amphiphilic block polymer in an organic solvent by coexisting the amphiphilic polymer and the organic solvent under heating. Heating activates the molecular motion of the polymer, thus promoting the swelling and dissolution of the amphiphilic polymer by the organic solvent. The heating temperature should be within the range of the boiling point of the solvent, for example, around 50 to 95°C, with 60 to 90°C being preferred. When the solution or swollen product of the amphiphilic block polymer cools below its gelation point, the formation of physical crosslinks in the hydrophobic block chains is promoted, resulting in an organogel with low fluidity (or no fluidity).

A xerogel (dry gel) can be obtained by removing the organic solvent used as a dispersion medium from the organogel. The method for removing the organic solvent from the organogel is not particularly limited and includes methods such as precipitating the gel by contact with a non-solvent, drying with a gas such as nitrogen, vacuum drying, heat drying, heat vacuum drying, freeze-drying, and supercritical drying. For purposes such as accelerating the removal of the organic solvent, the organogel may be pulverized into particles before removing the solvent. Alternatively, the gel may be pulverized while removing the solvent.

The degree to which the organic solvent is removed is not particularly limited, but it is preferable to remove the solvent until it becomes a solid that does not wet. It is preferable that the xerogel contains substantially no dispersion medium. The content of the dispersion medium in the xerogel is preferably 20% by weight or less, more preferably 10% by weight or less, even more preferably 5% by weight or less, and may be 1% by weight or less, based on the total amount of the gel composition. By sufficiently removing the organic solvent when forming the xerogel from the organogel, the content of the organic solvent in the hydrosol or hydrogel formed from the xerogel can be reduced, thereby lowering toxicity to cells and improving biosafety.

A hydrosol or hydrogel can be obtained by mixing xerogel and water. The hydrosol may contain gel-like portions. In xerogels from which the solvent has been removed from organogels, the physical cross-linking structure from the organogel formation is easily maintained, and the physical cross-linking structure is also easily maintained in hydrosols or hydrogels obtained by wetting the xerogel with water. The amount of residual organic solvent can also be reduced by wetting the xerogel with water.

The water used to wet the xerogel may be distilled water or an aqueous solution. Preferably, the aqueous solution is a liquid that does not damage cells, such as physiological saline, buffer solution, or culture medium. An aqueous solution containing suspended cells may be added to the xerogel to form a hydrosol or hydrogel. The hydrosol or hydrogel formed by adding cells and culture medium can be used directly for cell culture. In this case, there is no need to transplant cells into the hydrosol or hydrogel, and the cells can be dispersed within the gel composition, resulting in excellent workability.

The gel composition may contain various substances used in cell culture. Examples of substances used in cell culture include various inorganic salts, carbohydrates, amino acids, vitamins, fatty acids, lipids, proteins, peptides, etc. The gel composition may also contain cell adhesion epitopes, receptor agonists, receptor antagonists, ligands, extracellular matrix components, etc. Amphiphilic polymers may be modified with functional groups having these functions. The gel composition may also contain preservatives, plasticizers, surfactants, defoamers, stabilizers, buffers, pH adjusters, osmotic pressure adjusters, isotonic agents, etc.

In the preparation of hydrosols or hydrogels, the ratio of amphiphilic polymer to water is not particularly limited and should be set within a range that allows the gel to be wetted, depending on the molecular weight and mass of the amphiphilic polymer. From the viewpoint of appropriately maintaining the intermolecular distance between adjacent amphiphilic block polymers, the amount of water mixed is preferably 50 parts by weight or more and 1500 parts by weight or less, and more preferably 100 parts by weight or more and 1000 parts by weight or less, per 100 parts by weight of amphiphilic polymer. In hydrosols, the content of amphiphilic block polymer in the wetted gel composition is preferably 0.1% by weight or more. In hydrogels, the content of amphiphilic block polymer in the wetted gel composition is preferably 10% by weight or more.

After forming a hydrosol or hydrogel, water may be removed to form a xerogel. For example, if the gel composition contains substances insoluble in organic solvents or substances that are easily decomposed by organic solvents, these substances can be mixed into the hydrosol or hydrogel, and then water can be removed to obtain a xerogel containing these substances. The obtained xerogel can then be re-wetted with water to obtain a hydrosol or hydrogel.

From the viewpoint of reducing toxicity and irritation to living organisms, it is preferable that the hydrosol or hydrogel contains as little organic solvent as possible. The proportion of water in the total dispersion medium of the hydrosol or hydrogel is preferably 80% by weight or more, more preferably 90% by weight or more, even more preferably 95% by weight or more, and particularly preferably 98% by weight or more. To reduce the organic solvent content, it is preferable to increase the removal rate of organic solvents when forming xerogels from organogels. The organic solvent content can also be reduced by repeatedly forming a hydrosol or hydrogel and then removing the dispersion medium to form a xerogel after forming a xerogel from an organogel.

[Method for manufacturing cell culture vessels]
A method for manufacturing a cell culture vessel according to one embodiment of the present invention is:
The process includes the step of applying a gel composition to the recesses of a cell culture vessel having recesses. The gel composition comprises an amphiphilic block polymer having hydrophilic block chains and hydrophobic block chains.

The cell culture vessel preferably has a plurality of recesses. The recesses of the cell culture vessel preferably decrease in diameter from the opening towards the bottom. The recesses and gel composition of the cell culture vessel may be as described in the cell culture vessel section above. Each step of the manufacturing method according to the present invention can be followed by referring to the description of the cell culture vessel above. According to this manufacturing method, a cell culture vessel suitable for producing a uniform cell aggregate can be manufactured.

In the step of applying the gel composition to the recess, the gel composition in a fluid state can be dropped onto the cell culture area (e.g., well). The gel composition used for application may be an organogel, hydrosol, or hydrogel, but an organogel is preferred. By mixing the amphiphilic block polymer with an organic solvent, the amphiphilic block polymer self-assembles and takes on a rod-like (fibrous) structure. The concentration of the amphiphilic block polymer in the organogel may be, for example, 0.1 mg/mL or more and 200 mg/mL or less, and may be around 1 mg/mL, 2 mg/mL, 10 mg/mL, or 50 mg/mL.

The manufacturing method according to the present invention preferably includes a step of drying the coated gel composition. This step yields a cell culture vessel in which the xerogel, from which the dispersion medium has been removed, is laminated (fixed) in the recesses. The method for removing the dispersion medium is not particularly limited and may include precipitating the gel by contact with a non-solvent, drying with a gas such as nitrogen, vacuum drying, heat drying, heat vacuum drying, freeze-drying, supercritical drying, etc. If the dispersion medium is an organic solvent, removing the dispersion medium can reduce toxicity to cells. It is believed that the rod structure of the amphiphilic block polymer is maintained even after the dispersion medium is removed.

Xerogel can be stored at room temperature and does not require refrigeration or freezing. Therefore, cell culture containers containing the xerogel can also be stored at room temperature. In this form, a moistened gel composition is obtained by adding water to the xerogel during preparation, resulting in excellent handling.

In the step of applying the gel composition to a recess, it is preferable to apply the gel composition to a recess that has been pre-treated to be hydrophilic. The water-moistened gel composition can be separated from the surface of the recess. The hydrophilic treated recess exhibits cell non-adhesion, allowing for the stable acquisition of cell aggregates.

[Cell culture method]
A cell culture method relating to one embodiment of the present invention is
A cell culture method using a cell culture vessel,
A step of preparing a cell culture vessel having a recess, wherein a gel composition is applied to the recess,
The process includes a step of wetting the gel composition.

The cell culture vessel having a recess may be the cell culture vessel described above. Methods for preparing the cell culture vessel include, for example, purchasing a cell culture vessel with the recess pre-coated with a gel composition (at least a portion of the recess is covered with the gel composition), or applying the gel composition to the recess of an existing cell culture vessel.

The cell culture method according to the present invention includes a step of wetting a gel composition. The gel composition in the recesses can be wetted to form a three-dimensional network. Cells are cultured within this three-dimensional network, and preferably, cell aggregates are formed. According to the cell culture method according to the present invention, it is easier to produce cell aggregates of uniform size. Furthermore, the wetted gel composition increases the cohesive force of the cell aggregates, further assisting in the formation of cell aggregates. By culturing cell aggregates in the gel composition, larger cell aggregates can be obtained, or the properties of the cells can be brought closer to in vivo properties or desired properties.

The gel composition can be moistened with water; for example, water can be added to the depressions. The water may be an aqueous solution such as physiological saline, buffer solution, or culture medium. Cells may be added or embedded after moistening the xerogel gel composition, or cells and water may be added to the gel composition simultaneously. Moistening the gel composition by adding a culture medium in which cells are suspended to the depressions simplifies the work process and allows for uniform dispersion of cells.

The gel composition is preferably separated from the surface of the recesses after wetting. The gel composition can separate from the recesses to form a three-dimensional network. This makes the cells more likely to aggregate, and allows for easy acquisition of amphiphilic block polymers and cell aggregates separated from the recesses. Furthermore, the cell aggregates can be easily recovered from the cell culture vessel after culturing.

An example of a cell culture method according to the present invention is shown in Figure 2. (1) The cell culture vessel 10 is a dish, and its culture surface is provided with irregularities. The surface of the recesses 11 of the cell culture vessel is covered with a gel composition 12 which is a xerogel. (2) A culture medium is added to this cell culture vessel and cells 20 are seeded. (3) By adding water, the gel composition 12 is moistened. The amphiphilic block polymer separates from the surface of the cell culture vessel and forms a fibrous three-dimensional network. (4) The cells aggregate in the recesses, and aggregates 30 of uniform size are formed.

The type of cells to be cultured is not particularly limited. The cells may be, for example, animal cells, mammalian cells, non-human animal cells, or human cells. Examples of cells may include induced pluripotent stem cells, embryonic stem cells, stem cells, osteoblasts, fibroblasts, hematopoietic cells, antibody-producing cells, hepatocytes, spleen cells, kidney cells, gastrointestinal tract-derived cells, smooth muscle cells, cardiomyocytes, lung cells, nerve cells, adipocytes, and their precursor cells.

The number of cells seeded in each recess may be, for example, 50 or more or 1 × ^10² or more, and may be 1 × ^10⁶ or less. The number of cells constituting the cell aggregate after culture may be, for example, 50 or more, and may be 1 × ^10⁴ or less.

The cell culture conditions are not particularly limited, and general cell culture conditions can be applied. The cell culture period can be appropriately selected depending on the desired cell properties, for example, it may be between 6 hours and 100 days, or between 1 day and 2 days and 60 days or 30 days or less. After harvesting the cell aggregate, the culture may be continued further depending on its intended use, or it may be cultured further in combination with other cells.

The cultured cells may be recovered while still encapsulated within the three-dimensional network of the gel composition, or the three-dimensional network of the gel composition may be decomposed to recover the cultured cells. By adding water to the gel to reduce the concentration of the gel composition, the interactions between adjacent polymers are weakened, and the three-dimensional network of amphiphilic polymers is easily decomposed. For example, after culturing cells in a hydrosol, the cell aggregate can be recovered by adding water such as culture medium to the hydrosol. The cell aggregate can also be recovered by decomposing the three-dimensional network of the gel composition by pipetting and then centrifuging it.

The amphiphilic block polymer that forms the base of the gel composition is biodegradable and unlikely to exhibit toxicity even when transplanted into living organisms. Therefore, cultured cell aggregates containing the amphiphilic block polymer can be directly transplanted into living organisms. The cultured cells can also be used for drug screening and other applications.

[Experiment 1]
Referring to the method described in International Publication No. 2009/148121, sarcosine anhydride was polymerized to aminated poly-L-lactic acid in the presence of glycolic acid, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and N,N-diisopropylethylamine (DIEA) to synthesize a linear amphiphilic block polymer (PLA ₃₂ -PSar ₁₀₈ ) having a hydrophilic block consisting of 108 sarcosine units and a hydrophobic block consisting of 32 L-lactic acid units.

2 mg of amphiphilic block polymer was dissolved in 1 mL of 95% ethanol by heating to 70°C. This solution was added to one well of a 24-well plate (EZsphere plate with a textured bottom surface, made of polystyrene, AGC Technoglass Co., Ltd.). The plate was air-dried overnight in a safety cabinet to form a xerogel. The amount of residual ethanol was 0.04% by mass or less. A cell suspension was added to the well in which the xerogel had formed, and the cells were cultured at 37°C in a 5% _CO2 environment. Upon addition of the cell suspension, the xerogel absorbed water, and a hydrosol was formed. After culturing, it was confirmed that a uniform cell aggregate had been formed.

[Experiment 2]
A 24-well plate equipped with xerogel was prepared in the same manner as in Experiment 1, except that the amount of amphiphilic blocking polymer was adjusted to 50 mg/well. 500 μL of culture medium containing 5.0 × ^10⁵ HepG2 cells was added to the wells containing the xerogel. The cells were cultured at 37°C in a 5% _CO₂ environment for 24 hours. As a comparative example, wells from an EZsphere plate without a gel composition coating were used. Compared to the case without a gel composition coating, the wells coated with the gel composition were able to form uniform cell aggregates in the depressions (Figure 3). In the wells without a gel composition coating, a lot of cell spillage from the depressions was observed. Because the gel composition increases the cohesive force of the cell aggregates, there is less cell spillage and larger cell aggregates are obtained in the wells coated with the gel composition.

[Pattern]
Those skilled in the art will understand that the above-described exemplary embodiments and examples are specific examples of the following embodiments.

(Section 1)
A cell culture vessel according to one embodiment is:
A cell culture vessel having multiple recesses,
The aforementioned recess decreases in diameter from the opening towards the bottom.
The recess is covered with a gel composition containing an amphiphilic block polymer having hydrophilic block chains and hydrophobic block chains.

According to the cell culture vessel described in paragraph 1, it is easier to form cell aggregates of uniform size.
(Section 2)
In the cell culture vessel described in paragraph 1, the hydrophilic block chain contains sarcosine units, and the hydrophobic block chain contains lactate units.

According to the cell culture vessel described in paragraph 2, a hydrosol more suitable for cell culture can be formed.

(Section 3)
In the cell culture vessel described in paragraph 1 or 2, the gel composition is a xerogel.

In the cell culture vessel described in paragraph 3, the amphiphilic block polymer can be formed into a xerogel while maintaining a rod structure in which the hydrophobic block portions are aggregated. Since the xerogel is stable even at room temperature, the cell culture vessel containing the xerogel can also be stored at room temperature.

(Section 4)
In the cell culture vessel according to paragraph 3, the moistened gel composition is separable from the surface of the recess.

According to the cell culture vessel described in Section 4, the amphiphilic block polymer separated from the recess enhances the cohesive force of the cells, thus allowing for the more stable acquisition of cell aggregates.

(Section 5)
In the cell culture vessel described in any of paragraphs 1 to 4, the moistened gel composition has cell non-adhesion properties.

According to the cell culture vessel described in Section 5, the cohesive force of the cells is enhanced, making it possible to obtain cell aggregates more stably.

(Section 6)
In the cell culture vessel described in any of paragraphs 1 to 5, the cell culture vessel contains polystyrene resin.

The cell culture vessel described in paragraph 6 has excellent moldability and sterilization properties.
(Section 7)
In the cell culture vessel described in any of paragraphs 1 to 6, the gel composition is laminated on the surface of the hydrophilically treated recess.

The cell culture vessel described in paragraph 7 is treated with a hydrophilic coating, which prevents cells from adhering to the recessed areas. This facilitates cell aggregation and the formation of cell aggregates within the recessed areas, and also makes it easy to remove these cell aggregates.

(Section 8)
In a cell culture vessel described in any of paragraphs 1 to 6,
The cell culture vessel is a multi-well plate or dish.
The aforementioned recess is an uneven surface provided on the bottom of the portion that contains the culture medium.

(Section 9)
In a cell culture vessel described in any of paragraphs 1 to 6,
The cell culture vessel is a multi-well plate.
The vertical cross-sectional shape of the bottom of each well is conical, frustoconical, or hemispherical.

(Section 10)
A cell culture method relating to one embodiment is:
A cell culture method using a cell culture vessel,
A step of preparing a cell culture vessel having a recess, wherein a gel composition is applied to the recess,
The process includes a step of wetting the gel composition.

According to the cell culture method described in Section 10, it becomes easier to obtain cell aggregates of uniform size.
(Section 11)
In the cell culture method described in paragraph 10, the culture medium in which the cells are suspended is added to the recess to wet the gel composition.

According to the cell culture method described in paragraph 11, the work process can be simplified by adding the cells and culture medium simultaneously. By adding the culture medium in which the cells are suspended to the recesses coated with the gel composition, the gel composition is moistened, and the cells can be cultured in a three-dimensional network of swollen amphiphilic block polymers.

(Section 12)
In the cell culture method described in paragraph 10 or 11, the gel composition is separated from the surface of the recess after wetting.

According to the cell culture method described in paragraph 12, the amphiphilic block polymer constituting the gel composition enhances the cohesive force of the cells, allowing for the formation of more stable cell aggregates.

(Section 13)
A method for manufacturing a cell culture vessel according to one embodiment is:
The process includes the step of applying a gel composition to the recess of the cell culture vessel having a recess,
The gel composition comprises an amphiphilic block polymer having hydrophilic block chains and hydrophobic block chains.

According to the manufacturing method described in paragraph 13, a cell culture vessel can be obtained in which the size of the cell aggregate tends to be uniform.

(Section 14)
In the manufacturing method described in paragraph 13, the gel composition used for coating is an organogel.

According to the manufacturing method described in paragraph 14, a rod-shaped structure formed by the self-assembly of an amphiphilic block polymer can be constructed on the surface of the recess.

(Section 15)
The manufacturing method described in paragraph 13 or 14 includes a step of drying the coated gel composition.

According to the manufacturing method described in paragraph 15, the xerogel is formed on the surface of the recess. The xerogel can be stored at room temperature, and the cell culture container containing the xerogel can also be stored at room temperature.

(Section 16)
In the manufacturing method described in paragraphs 13 to 15, the gel composition is applied to the hydrophilically treated recess.

According to the manufacturing method described in paragraph 16, a cell culture vessel is obtained in which a gel composition is laminated on the surface of a recess that has become cell-non-adherent due to hydrophilic treatment. When the recess is cell-non-adherent, cells tend to aggregate inside the recess to form cell clumps, and the cell clumps can be easily removed.

10 Cell culture vessel, 11 Recess, 12 Gel composition, 20 Cells, 30 Cell aggregate.

Claims (18)

A cell culture vessel having multiple recesses,
The recessed portion tapers in diameter from the opening towards the bottom, and the major axis of the opening surface is 1500 μm or less.
A cell culture vessel in which the recess is covered with a gel composition comprising an amphiphilic block polymer having hydrophilic block chains containing 20 or more sarcosine units and hydrophobic block chains containing 10 or more lactate units . The cell culture vessel according to claim 1, wherein the gel composition is a xerogel. The cell culture vessel according to claim 2 , wherein the moistened gel composition is separable from the surface of the recess. The cell culture vessel according to claim 1, wherein the moistened gel composition has cell non-adhesion properties. The cell culture vessel according to claim 1, wherein the cell culture vessel comprises polystyrene resin. The cell culture vessel according to claim 1, wherein the gel composition is laminated on the surface of the hydrophilically treated recess. The cell culture vessel is a multi-well plate or dish.
The cell culture vessel according to claim 1, wherein the recess is an uneven surface provided on the bottom surface of the portion that contains the culture medium. The cell culture vessel is a multi-well plate.
The cell culture vessel according to claim 1, wherein the vertical cross-sectional shape of the bottom of each well is conical, frustoconical, or hemispherical. A cell culture vessel having multiple recesses,
The recessed portion tapers in diameter from the opening towards the bottom, and the bottom surface has irregularities formed by a continuous curved surface.
A cell culture vessel in which the recess is covered with a gel composition comprising an amphiphilic block polymer having hydrophilic block chains containing 20 or more sarcosine units and hydrophobic block chains containing 10 or more lactate units . A cell culture method using a cell culture vessel,
A step of preparing a cell culture vessel having a recess, wherein the major axis of the opening surface of the recess is 1500 μm or less, and a gel composition is applied to the recess, wherein the gel composition comprises an amphiphilic block polymer having hydrophilic block chains containing 20 or more sarcosine units and hydrophobic block chains containing 10 or more lactic acid units ,
A method comprising the step of wetting the gel composition. The cell culture method according to claim 10 , wherein a culture medium in which cells are suspended is added to the recess to wet the gel composition. The cell culture method according to claim 10 , wherein the gel composition is separated from the surface of the recess after wetting. A cell culture method using a cell culture vessel,
A step of preparing a cell culture vessel having a recess, the recess tapering in diameter from the opening towards the bottom, and the bottom surface having irregularities formed by a continuous curved surface, wherein a gel composition is applied to the recess , the gel composition comprising an amphiphilic block polymer having hydrophilic block chains containing 20 or more sarcosine units and hydrophobic block chains containing 10 or more lactic acid units ,
A method comprising the step of wetting the gel composition. A method for manufacturing a cell culture vessel,
The process includes applying a gel composition to the recess of a cell culture vessel having a recess, wherein the major axis of the opening surface of the recess is 1500 μm or less.
A method for producing the gel composition comprising an amphiphilic block polymer having a hydrophilic block chain containing 20 or more sarcosine units and a hydrophobic block chain containing 10 or more lactic acid units . The manufacturing method according to claim 14 , wherein the gel composition used for coating is an organogel. The manufacturing method according to claim 14 , further comprising the step of drying the coated gel composition. The manufacturing method according to claim 14 , wherein the gel composition is applied to the hydrophilically treated recess. A method for manufacturing a cell culture vessel,
The process includes applying a gel composition to the recess of a cell culture vessel having a recess, the recess being smaller in diameter from the opening towards the bottom, and the bottom surface having irregularities formed by a continuous curved surface,
A method for producing the gel composition comprising an amphiphilic block polymer having a hydrophilic block chain containing 20 or more sarcosine units and a hydrophobic block chain containing 10 or more lactic acid units .