JP7034467B2 - Cell three-dimensional culture method, cell structure, cell structure manufacturing method, cell transport method and cell preservation method - Google Patents

Description

The present invention relates to a three-dimensional cell culture method, a cell structure, a method for producing a cell structure, and a method for transporting cells.

The three-dimensional cell culture method is known as a method for culturing a large amount of cells. In particular, adhesive cells, which are the majority of cells in the body, do not proliferate at all when simply suspended in a medium and cultured. Therefore, it is common to carry out suspension culture together with a member called a microcarrier, which serves as a scaffold to which cells adhere.

Patent Documents 1 and 2 disclose a method for culturing a large amount of adhesive cells using microcarriers.

Japanese Unexamined Patent Publication No. 2011-030453 Japanese Unexamined Patent Publication No. 2017-136042

In the conventional suspension culture method using microcarriers described in Patent Documents 1 and 2, the microcarriers form large agglomerates via cells, and the permeability of oxygen and nutrients is reduced. It has become a problem that it leads to cell death inside the aggregate and change in cell properties.

The present invention has been made in view of the above circumstances, and provides a three-dimensional culture method for cells in which cell death due to excessive aggregation of microcarriers and cells is suppressed. Further, the present invention provides a cell structure in which the size of microcarriers and cell aggregates is controlled and the cell viability and cell proliferation rate are good, and a method for producing the same. Further, the present invention provides a method for transporting cells that can safely and easily transport cells while maintaining the cell viability.

That is, the present invention includes the following aspects.
The method for three-dimensional culture of cells according to the first aspect of the present invention is a second method in which a first solution containing a polysaccharide, a microcarrier, and an adhesive cell are mixed and a coagulant for the polysaccharide is contained. A gelling step of adding a solution to gel the polysaccharide to obtain a fibrous mixture containing the microcarriers, the adhesive cells and the gelled polysaccharide, and a third containing polyamino acids. A first coating step of coating a fibrous mixture containing the microcarriers, the adhesive cells and the gel-like polysaccharide with the polyamino acid by adding a solution, and solubilization of the gel-like polysaccharide. A removal step of adding a fourth solution containing the agent to remove the polysaccharide, and a culture step of culturing a fibrous mixture containing the microcarrier and the adhesive cells coated with the polyamino acid. It is a method to prepare.

In the method for three-dimensionally culturing cells according to the first aspect, the first solution and the second solution are further added after the first coating step and before the removal step. A second coating step may be provided in which the fiber-like mixture containing the microcarriers coated with the polyamino acid, the adhesive cells and the gel-like polysaccharide is further coated with the gel-like polysaccharide. ..

In the method for three-dimensional culture of cells according to the first aspect, the first solution may further contain an extracellular matrix.

The cell structure according to the second aspect of the present invention contains microcarriers and adhesive cells, and the mixture containing the microcarriers and the adhesive cells is coated with polyamino acids and is in the form of fibers .

In the cell structure according to the second aspect, the mixture may further contain a gel-like polysaccharide.

In the cell structure according to the second aspect, the mixture may further contain an extracellular matrix.

In the cell structure according to the second aspect, the mixture coated with the polyamino acid may be further coated with a gel-like polysaccharide.

The method for producing a cell structure according to a third aspect of the present invention is a second method in which a microcarrier, an adhesive cell, and a first solution containing a polysaccharide are mixed and a coagulant for the polysaccharide is contained. A gelling step of adding a solution to gel the polysaccharide to obtain a fibrous mixture containing the microcarriers, the adhesive cells and the gel-like polysaccharide, and a third containing polyamino acids. A method comprising a first coating step of adding a solution to coat a fibrous mixture containing the microcarriers, the adhesive cells and the gel-like polysaccharide with polyamino acids.

In the method for producing a cell structure according to the third aspect, after the first coating step, a fourth solution containing the solubilizer for the gel-like polysaccharide is further added to remove the polysaccharide. It may be provided with a removal step.

In the method for producing a cell structure according to the third aspect, the first solution and the second solution are further added after the first coating step and before the removal step. A second coating step may be provided in which the fibrous mixture containing the microcarriers coated with the polyamino acid, the adhesive cells and the gel-like polysaccharide is further coated with the gel-like polysaccharide. ..

In the method for producing a cell structure according to the third aspect, the first solution may further contain an extracellular matrix.

The method for transporting cells according to the fourth aspect of the present invention is a method using the cell structure according to the second aspect.
The cell preservation method according to the fifth aspect of the present invention includes a preservation step of preserving the cell structure obtained by the production method according to the third aspect at 4 ° C. or higher and 40 ° C. or lower.

According to the three-dimensional cell culture method of the above-described embodiment, cell death due to excessive aggregation of microcarriers and cells can be suppressed. According to the cell structure of the above aspect and the method for producing the same, the size of microcarriers and cell aggregates can be controlled, and the cell viability and cell proliferation rate can be improved. According to the cell transport method of the above aspect, cells can be safely and easily transported while maintaining the cell viability.

It is a schematic process diagram which shows an example of the three-dimensional culture method of the cell of this embodiment. 9 is a microscopic image of a mixture containing microcarriers, C2C12 cells and gelled alginic acid in Example 1. The scale bar is 500 μm. It is a microscope image of the cell structure (containing gel-like alginic acid) in Example 1. The scale bar is 500 μm. It is a fluorescence microscope image of the cell structure (containing gel-like alginic acid) and the cell structure (without gel-like alginic acid) on the first day of culture in Example 1. The scale bar is 250 μm. It is an image which observed the control group, the cell structure (containing gel-like alginic acid) and the cell structure (without gel-like alginic acid) on the 4th day of culture in Example 1. The upper part is a microscope image in a bright field, and the lower part is a fluorescence microscope image. The scale bar is 400 μm. It is a graph which shows the cell proliferation rate in the control group, the cell structure (containing gel-like alginic acid) and the cell structure (without gel-like alginic acid) on the 4th day of culture in Example 1. It is a fluorescence microscope image of the cell structure (gel-like alginic acid-free) in Example 2. The scale bar is 500 μm. 3 is an observation photograph of a T-shaped cell structure (containing gel-like alginic acid) in Example 3. 3 is a graph showing the cell proliferation rate of each subcultured cell in Example 4. 6 is a microscope image of cells before differentiation induction in Example 5. The scale bar is 500 μm. It is a microscope image of the cell after the differentiation induction in Example 5. The scale bar is 500 μm. It is an image which observed the control group 1 (Static MC), the control group 2 (Agitation MC), and the cell structure (gel-free alginic acid-free) (MC fiber) on the 1st and 4th days of culture in Example 6. .. The left and center are microscope images in the bright field, and the right is a fluorescence microscope image. The scale bar is 500 μm. It is a graph which shows the cell proliferation rate in the control group 1 (Static MC), the control group 2 (Agitation MC) and the cell structure (gel-like alginate-free) (MC fiber) on the 4th day of culture in Example 6. ..

<Three-dimensional culture method of cells>
The cell three-dimensional culture method of the present embodiment is a method including a gelation step, a first coating step, a removal step, and a culture step.

FIG. 1 is a schematic process diagram showing an example of a three-dimensional cell culture method of the cells of the present embodiment. Each step will be described in detail below with reference to FIG.

[Gelification process]
In the gelling step, first, the first solution 1a containing the polysaccharide, the microcarrier 2 and the adhesive cell 3 are mixed. At this time, it is preferable to stir so that the mixed solution becomes uniform. Then, a second solution 4 containing a coagulant for the polysaccharide is added to the mixed solution in a uniform state to gel the polysaccharide. As a method of adding the mixed solution to the second solution, for example, as shown in FIG. 1, a syringe or the like is filled with the mixed solution, and the mixed solution is discharged into a container containing the second solution using a syringe. The method can be mentioned. Alternatively, for example, a method of sending a mixed solution and a second solution to the microchannel and bringing them into contact with each other can be mentioned.

In the gelling step, the polysaccharide is brought into contact with the coagulant to form a hydrogel to obtain a solid mixture containing the microcarrier 2, the adhesive cells 3 and the gelled polysaccharide 1b. In this mixture, the microcarrier 2 and the adhesive cells 3 are kept uniformly dispersed in the gel-like polysaccharide 1b.

(First solution)
The polysaccharide contained in the first solution may be any polysaccharide that can form a hydrogel by adding a coagulant and can be solubilized by adding a solubilizer. Specific examples of the polysaccharide include, but are not limited to, carrageenan, LM (Low Methylester) pectin, xyloglucan, gellan gum, hyaluronic acid and its salt, alginic acid and its salt, and the like. These polysaccharides may be used alone or in combination of two or more.

The hyaluronic acid and alginate are preferably salts of hyaluronic acid and alginic acid and monovalent metal ions. Examples of the monovalent metal ion include, but are not limited to, sodium, potassium and the like.

Among them, the polysaccharide is preferably alginic acid or a salt thereof.

In addition, other polysaccharides such as agar, gelatin, agarose, starch, glycogen, heparin, and cellulose may be used in combination with the above polysaccharides. These other polysaccharides can be dissolved by heating and can be cooled to form a hydrogel.

The concentration of the polysaccharide in the first solution can be, for example, 0.5% by mass or more and 3% by mass or less, and for example, 1% by mass or more and 2% by mass or less.

The solvent for dissolving the polysaccharide may be an aqueous solvent. Specific examples of the aqueous solvent include buffers known in the art such as phosphate buffer, Tris hydrochloride buffer, citrate buffer, carbon dioxide buffer, borate buffer, succinate buffer, and acetate buffer. Examples include liquids.

The buffer solution may contain NaCl, a surfactant (eg, Tween 20, Triton X-100, etc.), or a preservative (eg, sodium azide, etc.), if necessary.

Specific examples of known buffers include, for example, Phosphate Buffered Saline (PBS), PBS-T (PBS-Tween 20), Tris Buffered Saline (TBS), and TBS. -T (TBS-Tween 20) and the like, and are not limited thereto.

Further, instead of the aqueous solvent, a medium for cell culture may be used, or a mixed solution of the aqueous solvent and the medium may be used. The medium may be a basic medium containing components necessary for cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins) and the like.

Examples of the basic medium include DMEM, Medium Essential Medium (MEM), RPMI-1640, Basic Medium Eagle (BME), Dulvecco's Modified Eagle's Medium / Nutrient Mix-12M (Nutrient Mix-Tur). Examples include, but are not limited to, Glassgo Medium Essential Medium (Grasgow MEM).

In addition, the medium may further contain serum or a growth factor.

Examples of the serum include, but are not limited to, FBS / FCS (Fetal Bovine / Calf Serum), NCS (Newborn Calf serum), CS (Calf Serum), HS (Horse Serum) and the like.

The concentration of serum contained in the medium may be, for example, 2% by mass or more and 10% by mass or less.

Examples of the growth factor include, but are not limited to, cell proliferation factors, cell adhesion factors, and the like.

More specifically, as the growth factor, for example, epithelial growth factor (EGF), acidic fiberblast growth factor (aFGF), basic fiberblast growth factor (basic fiberblast growth). : BFGF), insulin-like growth factor-1 (IGF-1), macrophage-developed growth factor (MDGF), platelet-derived growth factor (Platelet-derivated growth factor) ), Tumor angiogenesis factor (TAF) and the like. These growth factors may be contained alone or in combination of two or more.

The concentration of the growth factor contained in the medium is not particularly limited, and may be, for example, 1 ng / mL or more and 10 μg / mL or less.

The medium may further contain other additives such as hormones, antibiotics and the like exemplified below.

Examples of hormones contained in the medium include insulin, glucagon, triiodothyronine, corticosteroids and the like. These hormones may be contained alone or in combination of two or more.

The concentration of the hormone contained in the medium is not particularly limited, and may be, for example, 1 ng / mL or more and 10 μg / mL or less.

Examples of the antibiotic contained in the medium include those used for normal animal cell culture such as gentamicin, amphotericin, ampicillin, minomycin, kanamycin, penicillin, streptomycin, gentamicin, tylosin, and aureomycin. These antibiotics may be contained alone or in combination of two or more.

The concentration of the antibiotic contained in the medium is not particularly limited, and may be, for example, 0.1 μg / mL or more and 100 μg / mL or less.

In addition, the first solution may further contain an extracellular matrix. The extracellular matrix is reconstituted from, for example, collagen (type I, type II, type III, type V, type XI, etc.) and mouse EHS tumor extract (including type IV collagen, laminin, heparan sulfate proteoglycan, etc.). Examples thereof include, but are not limited to, a basement membrane component (trade name: Matrigel), glycosaminoglycan, hyaluronic acid, proteoglycan, and gelatin.

(Micro carrier)
As the microcarrier used in the gelation step, a known microcarrier generally used for cell culture can be used. The shape thereof is not particularly limited, and examples thereof include a spherical shape, a polygonal shape, a spherical shape, a conical shape, a pyramidal shape, a truncated cone shape, and a truncated cone shape. Above all, it is preferably spherical because it has a large surface area and cells can adhere evenly.

When the microcarrier is spherical, its particle size can be, for example, 10 μm or more and 500 μm or less, and for example, 100 μm or more and 300 μm or less.

Further, the material of the microcarrier may be any material having cell compatibility. Specifically, the microcarrier may contain, for example, cellulose, dextran, hydroxylated methacrylate, hyaluronic acid, collagen, gelatin, polystyrene, plastic, glass, ceramic, silicone and the like. These may be contained alone or in combination of two or more.

When the material of the microcarrier is cellulose, dextran, hyaluronic acid, collagen, or gelatin, an enzyme (cellulase, dextranase) that decomposes the material of the microcarrier after reaching a desired number of cells in the culture step described later. Microcarriers can be easily removed by treatment with (nase, hyaluronidase, collagenase, protease) or the like. Furthermore, cells can be easily isolated by removing the polyamino acid that coats the cell structure. At this time, the cells are continuously cultured without removing the polyamino acid that coats the cell structure, and the cells are further proliferated so as to fill the space where the microcarriers have been removed to remove the polyamino acid. You can also. This makes it possible to obtain a cell structure consisting only of cells formed three-dimensionally.

The concentration of the microcarriers in the mixed solution of the first solution containing the polysaccharide, the microcarriers, and the adhesive cells can be, for example, 20 mg / mL or more and 100 mg / mL or less, for example, 30 mg / mL or more. It can be 80 mg / mL or less.

(Adhesive cells)
The adhesive cells used in the gelation step are not particularly limited as long as they adhere to the solid surface and proliferate. Specific examples of the adhesive cells include epithelial cells (Vero cells, MDCK cells, CHO cells, HEK293 cells, COS cells, HMLU cells, etc.), tumor cells (Hela cells, VACO cells, etc.), and endothelial cells (HUVE C cells). , DBAE cells, etc.), leukocytes (HIT-T15 cells, etc.), fibroblasts (WI38 cells, BHK21 cells, SFME cells, etc.), muscle cells (HL1 cells, C2C12 cells, etc.), nerve / endocrine gland cells (ROC-1 cells, etc.) , IMR-32 cells, etc.) and the like. These cells may be used alone or in combination of two or more.

The animal from which the cells are derived may be a vertebrate or an invertebrate. The vertebrate is not particularly limited, and examples thereof include mammals, amphibians, reptiles, amphibians, and fish. The invertebrate is not particularly limited, and examples thereof include insects, crustaceans, mollusks, and protozoa.

Among them, the animal from which the cells are derived is preferably a vertebrate, more preferably a mammal. Specific examples of mammals include, but are not limited to, humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, rats and the like. Above all, as a mammal, it is preferable that it is a human.

(Second solution)
The coagulant for the polysaccharide contained in the second solution may be appropriately selected depending on the type of the polysaccharide.
When the polysaccharide is carrageenan, LM (Low Methylester) pectin, gellan gum, alginic acid and a salt thereof, specific examples of the coagulant include a divalent metal salt. Examples of the divalent metal salt include, but are not limited to, barium salt, calcium salt, magnesium salt and the like.

Specific examples of the barium salt include barium chloride, barium fluoride, barium bromide, barium hydroxide, barium carbonate, barium phosphate and the like.

Calcium chloride, calcium fluoride, calcium bromide, calcium peroxide, calcium hydroxide, calcium carbonate, calcium nitrate, calcium sulfate, calcium phosphate and the like can be mentioned.

Examples thereof include magnesium chloride, magnesium fluoride, magnesium bromide, magnesium peroxide, magnesium hydroxide, magnesium carbonate, magnesium nitrate, magnesium sulfate, magnesium phosphate and the like.

When the polysaccharide is xyloglucan, specific examples of the coagulant include monosaccharides, oligosaccharides, alcohols, polyols, polyphenols and the like.

When the polysaccharide is hyaluronic acid, specific examples of the coagulant include aldehydes such as glutaraldehyde and glycerin aldehyde.

When the coagulant is a divalent metal salt, the concentration of the coagulant in the second solution can be, for example, 50 mM or more and 200 mM or less, for example, 75 mM or more and 150 mM or less.

The solvent for dissolving the coagulant may be an aqueous solvent. Examples of the aqueous solvent include the same as those exemplified in the above first solution. Further, instead of the aqueous solvent, a medium for cell culture may be used, or a mixed solution of the aqueous solvent and the medium may be used. Examples of the medium include the same as those exemplified in the above first solution.

[First coating step]
In the first coating step, a third solution 5 containing the polyamino acid is added to coat the mixture containing the microcarrier 2, the adhesive cells 3 and the gelled polysaccharide 1b with the polyamino acid 6. As a method of adding the third solution 5 to the mixture, for example, as shown in FIG. 1, a method of immersing the mixture in a heated third solution can be mentioned. Alternatively, for example, a method of dropping a warmed third solution onto the mixture can be mentioned. The temperature of the third solution can be, for example, 30 ° C. or higher and 40 ° C. or lower. As a result, a mixture containing microcarriers 2 coated with polyamino acids, adhesive cells 3 and the gel-like polysaccharide 1b (hereinafter referred to as "cell structure (containing gel-like polysaccharide)" 10) is obtained. Be done. Further, by coating with a polyamino acid, it is possible to prevent the mixture containing the microcarrier 2, the adhesive cell 3 and the gel-like polysaccharide 1b from aggregating with each other, and the microcarrier 2 and the adhesive cell contained in the mixture can be prevented from aggregating. It is possible to prevent 3 from spreading.

(Third solution)
Examples of the polyamino acid contained in the third solution include poly-L-ornithine, poly-L-lysine, poly-L-arginine, poly-L-histidine, poly-L-glutamic acid, and poly-L-aspartic acid. Etc., and are not limited to these. These polyamino acids may be used alone or in combination of two or more.

Among them, the polyamino acid is preferably poly-L-lysine.

When the coagulant is a divalent metal salt, the concentration of the polyamino acid in the third solution can be, for example, 50 mM or more and 200 mM or less, for example, 75 mM or more and 150 mM or less.

The solvent for dissolving the polyamino acid may be an aqueous solvent. Examples of the aqueous solvent include the same as those exemplified in the above first solution. Further, instead of the aqueous solvent, a medium for cell culture may be used, or a mixed solution of the aqueous solvent and the medium may be used. Examples of the medium include the same as those exemplified in the above first solution.

[Removal process]
In the removal step, a fourth solution 7 containing a solubilizing agent for the gel-like polysaccharide is added to the cell structure (containing the gel-like polysaccharide) 10 to remove the polysaccharide. As a method of adding the fourth solution, for example, as shown in FIG. 1, a method of immersing the cell structure (containing a gel-like polysaccharide) 10 in the fourth solution 7 can be mentioned. Alternatively, for example, a method of dropping the fourth solution 7 onto the cell structure (containing a gel-like polysaccharide) 10 can be mentioned. As a result, the gel-like polysaccharide contained in the cell structure (containing the gel-like polysaccharide) 10 is decomposed and removed to obtain the cell structure (without the gel-like polysaccharide).

(4th solution)
The solubilizing agent for the gel-like polysaccharide contained in the fourth solution may be appropriately selected depending on the type of the polysaccharide.

When the polysaccharide is carrageenan, LM (Low Methylester) pectin, gellan gum, and alginic acid and a salt thereof, examples of the solubilizer for the gel-like polysaccharide include a chelating agent and an enzyme.

Specific examples of the chelating agent include citric acid, ethylenediamine (Ethylenediamine), ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic Acid (NTA), and diethylenetriaminetetraacetic acid (Diethylenetriaminetetraacetic acid). DTPA), N- (2-Hydroxyethyl) Ethylenediamine-N, N', N'-Triacetic Acid (HydroxhylyEthylene Diamine Triacetic Acid; HEAD), Glycol Ether Diamine Tetracic Acid Triethylenetetramine-N, N, N', N'', N''', N'''-hexaacetic Acid (TTHA), N- (2-hydroxyethyl) iminodiacetic acid (Hydroxyethyl Imino) Examples thereof include, but are not limited to, ylenediamine Acid (HIDA), N, N-di (2-hydroxyethyl) glycine (Dihydroxyethyl Glycine; DHEG), and the like. Further, citric acid may be in the form of citrate such as sodium citrate and potassium citrate.

Examples of the enzyme include, but are not limited to, λ-carrageinase, pectinase, gellan gum degrading enzyme, alginate lyase and the like.

When the polysaccharide is xyloglucan and hyaluronic acid and a salt thereof, examples of the solubilizer for the gel-like polysaccharide include enzymes such as xyloglucan degrading enzyme and hyaluronidase.

When the solubilizer for the gel-like polysaccharide contained in the fourth solution is citric acid, the concentration of the citric acid can be, for example, 0.1 mM or more and 100 mM or less.

When the solubilizer for the gel-like polysaccharide contained in the fourth solution is EDTA, the concentration of the EDTA can be, for example, 0.5 mM or more and 100 mM or less.

When the solubilizer for the gel-like polysaccharide contained in the fourth solution is an enzyme, the concentration of the enzyme can be, for example, 0.01 mg / mL or more and 1 mg / mL or less. It can be 02 mg / mL or more and 500 mg / mL or less.

The solvent for dissolving the solubilizer for the gel-like polysaccharide may be an aqueous solvent. Examples of the aqueous solvent include the same as those exemplified in the above first solution. Further, instead of the aqueous solvent, a medium for cell culture may be used, or a mixed solution of the aqueous solvent and the medium may be used. Examples of the medium include the same as those exemplified in the above first solution.

[Culture process]
In the culturing step, a mixture containing microcarriers 2 and adhesive cells 3 coated with polyamino acid 6 (hereinafter, may be referred to as “cell structure (without gel-like polysaccharide)” 20) is cultivated. Examples of the culturing method include, as shown in FIG. 1, a method in which a cell structure (gel-free polysaccharide-free) 20 is placed in a spinner flask 8 containing a medium 9 and cultured. Alternatively, a method known as another three-dimensional culture method may be used. This makes it possible to proliferate the adherent cells in the cell structure (without gel-like polysaccharide) 20 so as to fill the space where the gel-like polysaccharide removed in the removal step was present. In addition, the presence of the gel-like polysaccharide keeps the microcarrier 2 and the adhesive cells 3 uniformly dispersed, thereby suppressing excessive aggregation even after the gel-like polysaccharide is removed. And the cells existing inside the cell structure are also sufficiently supplied with nutrients and oxygen. As a result, the three-dimensional culture method of the present embodiment has better cell survival rate and cell proliferation rate than the conventional three-dimensional culture method.

Further, as the medium used in the culturing step, the same medium as those exemplified in the above-mentioned first solution can be mentioned.

The culture conditions can be appropriately adjusted according to the type of cells contained in the cell structure 20. The culture temperature can be, for example, 30 ° C. or higher and 40 ° C. or lower. The culture environment can be, for example, air conditions containing about 5% CO ₂ .

Further, the cell three-dimensional culture method of the present embodiment may further include other steps in addition to the gelation step, the first coating step, the removal step, and the culture step. Examples of other steps include a second coating step, a recovery step, a cleaning step, and the like. The second coating step may be performed after the first coating step and before the removing step. The recovery step may be performed after the above-mentioned culture step. The cleaning step may be performed after each of the above steps and before the subsequent steps.

[Second coating step]
In the second coating step, the cell structure (containing gel-like polysaccharide) 10 obtained in the first coating step contains a first solution 1a containing a polysaccharide and a second coagulant for the polysaccharide. Add solution 4. As a method of adding the first solution 1a and the second solution 4, for example, a suspension of the cell structure (containing gel-like polysaccharide) 10 and the first solution 1a is prepared, and the suspension is prepared. A method of sending the solution 4 and the second solution 4 to the microchannel and bringing them into contact with each other can be mentioned.

As a result, a mixture (cell structure (containing gel-like polysaccharide) 10) containing microcarrier 2, adhesive cells 3 and gel-like polysaccharide 1b coated with polyamino acid 6 is further added to gel-like polysaccharide. It can be coated with 1b. The structure of the obtained cell structure is such that the outermost surface is coated with a gel-like polysaccharide 1b, the intermediate layer is a polyamino acid 6, and the inside thereof is a microcarrier 2, an adhesive cell 3 and a gel-like polysaccharide. There is a mixture containing 1b. The obtained cell structure may be referred to as "cell structure (containing gel-like polysaccharide inside and outside)" below.

[Recovery process]
In the recovery step, the adhesive cells 3 are recovered from the cell structure (without gel-like polysaccharide) 20 after culturing obtained in the culture step. Specifically, first, the polyamino acid is physically peeled off, and the mixture containing the microcarrier 2 and the adhesive cell 3 is taken out from the inside. Next, the microcarrier 2 and the adhesive cell 3 are dissociated by trypsin treatment or the like, and the adhesive cell 3 is recovered.

[Washing process]
In the washing step, the mixture containing the microcarrier 2, the adhesive cells 3 and the gelled polysaccharide 1b obtained in the gelation step may be washed. Alternatively, it may be performed to wash the cell structure (containing gel-like polysaccharide) 10 obtained in the first coating step. Alternatively, it may be performed to wash the cell structure (containing gel-like polysaccharide inside and outside) obtained in the second coating step. It may be performed to wash the cell structure (gel-like polysaccharide-free) 20 obtained in the above removal step. Alternatively, it may be carried out to wash the cell structure (without gel-like polysaccharide) 20 after culturing obtained in the culturing step.

In the washing step, the product obtained in the above step can be washed once or more (for example, about 2 times or more and 3 times or less) using an aqueous solvent or a medium. Examples of the aqueous solvent or medium used in the washing step include the same as those exemplified in the first solution.

<Cell structure>
The cell structure of this embodiment is obtained by the above-mentioned three-dimensional culture method for cells. In the schematic configuration diagram showing an example of the three-dimensional cell culture method of the cells of the present embodiment of FIG. 1, the configuration diagram of the cell structure is shown.

The cell structure (gel-free polysaccharide-free) 20 shown in FIG. 1 contains microcarrier 2 and adhesive cells 3. Also, the mixture containing microcarriers 2 and adhesive cells 3 is coated with polyamino acid 6. This cell structure (without gel-like polysaccharide) 20 has a good survival rate and proliferation rate of the adhesive cell 3. Therefore, it is suitably used for mass culture of adhesive cells 3.

The mixture containing the microcarrier 2 and the adhesive cell 3 may further contain an extracellular matrix. This allows the adherent cells to grow better. Examples of the extracellular matrix include the same as those exemplified in the above first solution.

Further, in the cell structure (containing gel-like polysaccharide) 10 shown in FIG. 1, a mixture containing microcarrier 2, adhesive cells 3 and gel-like polysaccharide 1b is coated with polyamino acid 6. That is, the cell structure of the present embodiment may further contain the gel-like polysaccharide 1b. In this cell structure (containing gel-like polysaccharide) 10, the proliferation rate of the adhesive cells 3 is low, but the survival rate is good. Therefore, it is suitably used for transporting adhesive cells.

Further, although not shown, in the cell structure (containing gel-like polysaccharide inside and outside) obtained by the second coating step of the above-mentioned three-dimensional culture method for cells, the outermost surface is gel-like polysaccharide 1b. The intermediate layer is coated with polyamino acid 6, and a mixture containing microcarrier 2, adhesive cells 3 and gel-like polysaccharide 1b is present therein. That is, in the cell structure of the present embodiment, the cell structure (containing the gel-like polysaccharide) 10 may be further coated with the gel-like polysaccharide 1b. This cell structure (containing gel-like polysaccharides inside and outside) has a low proliferation rate of adhesive cells 3 but a good survival rate. Therefore, it is suitably used for transporting adhesive cells.

The shape of the cell structure of the present embodiment is not particularly limited, and examples thereof include, and are not limited to, a sheet shape (plate shape), a fiber shape (fibrous shape), and a spherical shape. Among them, the shape is preferably fibrous (fibrous) because it is easy to manufacture. In particular, in the case of a fiber-like structure having a fine structure, cell death due to excessive aggregation of microcarriers and cells can be suppressed more effectively.

The size of the cell structure of the present embodiment may be any size as long as it can be cultured with stirring.
When the shape of the cell structure of the present embodiment is fibrous (fibrous), the tube diameter thereof can be, for example, 100 μm or more and 1000 μm or less. The length thereof can be, for example, 500 μm or more and 100 μm or less.

<Manufacturing method of cell structure>
The method for producing a cell structure of the present embodiment is a method including the gelation step and the first coating step.

According to the method for producing a cell structure of the present embodiment, a cell structure (containing a gel-like polysaccharide) 10 can be obtained.

The method for producing a cell structure of the present embodiment may further include the removal step, the culture step, and the second coating step in addition to the gelling step and the first coating step. The removal step may be performed after the first coating step. The culture step may be performed after the removal step. Further, the second coating step may be performed after the first coating step and before the removing step.

In the method for producing a cell structure of the present embodiment, the cell structure (gel-like polysaccharide-free) 20 can be obtained by providing the above-mentioned removal step.

By providing the above-mentioned culture step, the method for producing a cell structure of the present embodiment can proliferate the adhesive cells 3 in the cell structure (gel-like polysaccharide-free) 20.

The method for producing a cell structure of the present embodiment includes the above-mentioned second coating step, whereby a cell structure (containing gel-like polysaccharide inside and outside) can be obtained.

The method for producing a cell structure of the present embodiment may further include other steps in addition to the gelling step, the first coating step, the removing step, and the second coating step. Examples of the other steps include a washing step, a second removing step, and the like. The washing step is the same as that described in the above-mentioned three-dimensional culture method for cells. Moreover, the second removal step may be performed after the above-mentioned culture step.

[Second removal step]
In the second removal step, when the material of the microcarrier is cellulose, dextran, hyaluronic acid, collagen, or gelatin, the microcarrier is removed. Specifically, first, in the above culture step, after reaching a desired number of cells, the cells are treated with an enzyme (cellulase, dextranase, hyaluronidase, collagenase, protease) that decomposes the material of the microcarrier. Thereby, the microcarriers can be easily removed, and the cell structure is coated with a polyamino acid, does not contain microcarriers, and contains adhesive cells (hereinafter, "cell structure (microcarrier-free)"). May be referred to as) can be obtained.

Further, by further culturing the obtained cell structure (without microcarriers), the cells can be further proliferated so as to fill the space where the microcarriers have been removed. Then, by physically peeling off the polyamino acid and removing it, a cell structure of three-dimensionally formed adhesive cells can be obtained.

<How to transport cells>
The cell transport method of the present embodiment is a method using the above cell structure.

The cell structure used in the cell transport method of the present embodiment is preferably a cell structure (containing a gel-like polysaccharide) or a cell structure (containing a gel-like polysaccharide inside and outside). As shown in Examples described later, the cell structure (containing gel-like polysaccharide) has a low proliferation rate of the adhesive cells 3 but a good survival rate. Therefore, it is suitably used for transporting adhesive cells.

Examples of the transport method include a method in which the cell structure is enclosed in an openable and closable sealed container containing a medium and then transported.

Examples of the medium include the same as those exemplified in the above first solution.

Examples of the sealed container include, but are not limited to, a conical tube with a screw cap, a flask for cell culture with a screw cap, and the like.

The conditions for carrying can be appropriately selected depending on the type of cells to be carried.

The temperature during transportation can be, for example, 4 ° C. or higher and 40 ° C. or lower, for example, 10 ° C. or higher and 39 ° C. or lower, and for example, 18 ° C. or higher and 37 ° C. or lower.

Further, the environment during transportation may be, for example, under the condition of air containing about 5% CO ₂ .

The transportable time varies depending on the cell type, the number of cells, and the like. For example, it can be 1 hour or more and 300 days or less, for example, 1 day or more and 100 days or less, and for example, 3 days or more and 60 days or less.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the following Examples.

[Example 1]
1. 1. Preparation of cell structure (1) Gelling step A solution containing 2 mg / mL native collagen and 1% alginic acid was prepared. Next, microcarriers (final concentration: 60 mg / mL, trade name: Cytodex3, manufactured by GE Healthcare Japan) and mouse myoblast line C2C12 cells (final concentration: 1.0 × 10 ⁶ cells / mL) were added to the solution. ) Was mixed to prepare a mixed solution. Then, the mixed solution was filled in a syringe and discharged into a 100 mM calcium chloride solution to gel alginic acid. This gave a mixture containing microcarriers, C2C12 cells and gelled alginic acid (see FIG. 2A). In FIG. 2A, the arrows indicate cells.

(2) Coating Step Next, the mixture was placed in a polylysine solution and heated to 37 ° C. to coat polylysine. As a result, a cell structure containing polylysine-coated microcarriers, C2C12 cells and gel-like alginic acid (hereinafter, may be referred to as "cell structure (containing gel-like alginic acid)") was obtained (Fig.). See 2B). In FIG. 2B, the arrows indicate cells.

(3) Removal Step Next, the cell structure (containing gel-like alginic acid) obtained in (2) was cultured for 1 day using a medium containing or not containing 40 μg / mL alginate lyase. As the medium, Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) was used. The culture conditions were 37 ° C. and a 5% CO ₂ environment. In addition, stirring culture was performed using an orbital shaker (rotation speed: about 50 rpm). Then, each cell structure after culturing was stained with calcein AM (staining live cells). The results are shown in FIG. In FIG. 3, "Alginate lyase (-)" indicates a cell structure (containing gel-like alginic acid), and "Alginate lyase (+)" indicates a cell structure (without gel-like alginic acid).

From FIG. 3, in the cell structure (gel-free alginic acid-free) cultured using the alginic acid lyase-containing medium, the gel-like alginic acid was decomposed and removed. Therefore, C2C12 cells adhered to the surface of the microcarrier. On the other hand, in the cell structure (containing gel-like alginic acid) cultured using the alginate lyase-free medium, the C2C12 cells did not adhere to the microcarriers.

(4) Culturing step Next, the cells were replaced with DMEM (containing 10% FBS) free of alginate lyase, and the cell structure (containing gel-like alginic acid) and the cell structure (containing gel-like alginic acid) were added for another 3 days. The cells were cultured (4 days from the start of the culture).

In addition, as a control group, C2C12 cells (2.0 × 10 ⁵ cells / mL) are seeded in a 6-well plate and contain 4 mg / mL microcarriers (trade name: Cytodex3, manufactured by GE Healthcare Japan). The cells were stirred and cultured for 4 days using DMEM (containing 10% FBS). The culture conditions were 37 ° C. and a 5% CO ₂ environment. In addition, stirring culture was performed using an orbital shaker (rotation speed: about 50 rpm).

After culturing for 4 days, the cell structures (containing gel-like alginate), cell structures (without gel-like alginate) and the control group were subjected to calcein AM (staining live cells) and ethidium homodimer (staining dead cells). Was stained with. The results are shown in FIG. 4A. In FIG. 4A, a control group is shown with "MC suspension (conventional)". "MC fiber Alginate lyase (-)" indicates a cell structure (containing gel-like alginic acid). The “MC fiber Alginate lyase (+)” indicates a cell structure (gel-like alginate-free). The upper part is a microscope image in a bright field, and the lower part is a fluorescence microscope image. The scale bar is 400 μm.

From FIG. 4A, many dead cells were observed in the control group. On the other hand, the cell structure (containing gel-like alginic acid) and the cell structure (without gel-like alginic acid) were mostly living cells. It was speculated that this was because the size of microcarriers and cell aggregates was controlled in the cell structure, and excessive aggregation was suppressed.

The cell structure (containing gel-like alginate), the cell structure (without gel-like alginate) and the control group were then treated with trypsin-EDTA to dissociate the cells and microcarriers. Then, the number of cells was measured. The results are shown in FIG. 4B. In FIG. 4B, the cell proliferation rate shows a relative ratio when the number of cells at the start of culture is 1. In FIG. 4B, a control group is shown with "MC suspension (conventional)". "MC fiber Alginate lyase (-)" indicates a cell structure (containing gel-like alginic acid). The “MC fiber Alginate lyase (+)” indicates a cell structure (gel-like alginate-free).

From FIG. 4B, cells hardly proliferated in the cell structure (containing gel-like alginic acid). On the other hand, in the control group, the cell proliferation rate was 6.9 times, whereas in the cell structure (gel-like alginic acid-free), the cell proliferation rate was 9.9 times, showing a high cell proliferation rate.

[Example 2]
1. 1. Preparation of Cell Structure A cell structure (containing gel-like alginic acid) was obtained by using the same method as in (1) to (2) of "1." of Example 1.

2. 2. Cultured cell structures (containing gel-like alginic acid) were cultured in DMEM (containing 10% FBS) for 2 days.

3. 3. Removal of gel-like alginic acid Next, the cells were replaced with DMEM containing 40 μg / mL alginate lyase, and the cell structure (containing gel-like alginic acid) cultured in “2.” for 2 days was cultured for 1 day, and the cell structure (gel) was cultured. Alginic acid-free) was obtained. As the culture conditions, an orbital shaker (rotational speed: about 50 rpm) was used for stirring and culturing. The cultured cell structures were then stained with calcein AM (staining live cells). The results are shown in FIG. In FIG. 5, the scale bar is 500 μm.

From FIG. 5, it was observed that the cells adhered onto the microcarriers and took a flat shape. Most of them were living cells.

From this, it was suggested that the cells could be transported in a cell structure not treated with alginate lyase (containing gel-like alginate), treated with alginate lyase at any timing, and the culture could be resumed.

[Example 3]
1. 1. Preparation of cell structure First, a cell trainer (pore: 70 μm) was immersed in a 100 mM calcium chloride solution and taken out. A solution containing 2 mg / mL native collagen and 1% alginic acid was then prepared. Next, microcarriers (final concentration: 60 mg / mL, trade name: Cytodex3, manufactured by GE Healthcare Japan) and mouse myoblast line C2C12 cells (final concentration: 1.0 × 10 ⁶ cells / mL) were added to the solution. ) Was mixed to prepare a mixed solution. Then, this mixed solution was dropped in a T shape onto a cell trainer infiltrated with a calcium chloride solution using a pipette. As a result, the portion in contact with the cell trainer infiltrated with the calcium chloride solution gelled, and a T-shaped cell structure was obtained (see FIG. 6). Also, since the pore size of the cell trainer does not allow microcarriers to pass through, cells and microcarriers were not lost.

From this result, it was suggested that a cell structure having an arbitrary three-dimensional structure could be obtained.

[Example 4]
1. 1. Preparation of Cell Structure A cell structure (gel-free alginic acid-free) was obtained by using the same method as in (1) to (4) of "1." of Example 1.

2. 2. Recovery Step Next, the polylysine of the cell structure (gel-free alginic acid-free) obtained in "1." was physically peeled off, and a mixture containing microcarriers and cells was taken out from the inside. The cells were then recovered by dissociating the microcarriers from the cells by trypsin-EDTA treatment. The obtained cells were designated as the first passage (Passage 1; P1).

3. 3. Preparation of cell structure (second time) and recovery process (second time)
Then, using the obtained P1 cells, a cell structure (gel-free alginic acid-free) was obtained by the same method as in (1) to (4) of "1." of Example 1. .. Then, using the obtained cell structure (gel-free alginic acid-free), cells were recovered by the same method as in "2." above. The obtained cells were designated as the second passage (Passage 2; P2).

4. Preparation of cell structure (3rd time) and recovery process (3rd time)
Then, using the obtained P2 cells, a cell structure (gel-free alginic acid-free) was obtained by the same method as in (1) to (4) of "1." of Example 1. .. Then, using the obtained cell structure (gel-free alginic acid-free), cells were recovered by the same method as in "2." above. The obtained cells were designated as the third passage (Passage 3; P3).

5. Confirmation of cell proliferation rate P1, P2 and P3 when the number of cells of P1, P2 and P3 was measured and the number of cells used for the initial culture (2 × 10 ⁶ cells / per cell structure) was 1. The ratio of the number of each cell was calculated as the cell proliferation rate. The results are shown in FIG.

From FIG. 7, it was confirmed that the cells proliferated stably.

From this result, it was suggested that the cell structure of the present embodiment can be used as a means for continuously proliferating cells.

[Example 5]
1. 1. Preparation of Cell Structure A cell structure (gel-free alginic acid-free) was obtained by using the same method as in (1) to (4) of "1." of Example 1.

2. 2. Recovery Step Next, using the cell structure (gel-free alginic acid-free) obtained in "1.", cells were recovered in the same manner as in "2." of Example 4.

3. 3. Differentiation induction Next, the recovered cells were seeded and cultured for 4 days using 2% by mass horse serum-containing DMEM to induce differentiation of C2C12 cells into myotube cells. In addition, as a control group, the collected cells were cultured in DMEM for 3 days. A microscopic image of the control group (cells before differentiation induction) is shown in FIG. 8A, and the cells after differentiation induction are shown in FIG. 8B. In FIGS. 8A and 8B, the scale bar is 500 μm.

From FIGS. 8A and 8B, it was confirmed that the cells were fused with each other by the induction of differentiation to form thick, multinucleated myotube cells.

This suggests that the three-dimensional culture method of the present embodiment retains the properties of cells.

[Example 6]
1. 1. Preparation of Cell Structure A cell structure (gel-free alginic acid-free) was obtained by using the same method as in (1) to (4) of "1." of Example 1.

In addition, as control group 1, C2C12 cells ⁽ 2.0 × 105 cells / mL) were seeded in a 6-well plate and contained 4 mg / mL microcarriers (trade name: Cytodex3, manufactured by GE Healthcare Japan). The cells were statically cultured for 4 days using DMEM (containing 10% FBS). The culture conditions were 37 ° C. and a 5% CO ₂ environment.

Furthermore, as control group 2, C2C12 cells ⁽ 2.0 × 105 cells / mL) were seeded in a 6-well plate and contained 4 mg / mL microcarriers (trade name: Cytodex3, manufactured by GE Healthcare Japan). DMEM (containing 10% FBS) was used for stirring and culturing for 4 days. The culture conditions were 37 ° C. and a 5% CO ₂ environment. In addition, stirring culture was performed using an orbital shaker (rotation speed: about 50 rpm).

After culturing for 4 days, cell structures (containing gel-like alginic acid), control group 1 and control group 2 were stained with calcein AM (staining live cells) and ethidium homodimer (staining dead cells). The results are shown in FIG. 9A. In FIG. 9A, “MC fiber” refers to a cell structure (containing gel-like alginic acid). “Static MC” refers to control group 1. “Agitation MC” refers to control group 2. The left and center are microscope images in the bright field, and the right is a fluorescence microscope image. The scale bar is 500 μm.

From FIG. 9A, in the cell structure (containing gel-like alginic acid), the number of living cells was significantly higher than that of dead cells.
On the other hand, in control group 1, cell aggregation could not be controlled, and many dead cells were observed inside. Furthermore, in the control group 2, although relatively small agglomerates were formed, a large amount of dead cells were observed due to shear stress (shear stress of the fluid) due to stirring.

The cell structure (containing gel-like alginic acid), control group 1 and control group 2 were then treated with trypsin-EDTA to dissociate the cells and microcarriers. Then, the number of cells was measured. The results are shown in FIG. 9B. In FIG. 9B, the cell proliferation rate shows a relative ratio when the number of cells at the start of culture is 1. In FIG. 9B, “MC fiber” refers to a cell structure (containing gel-like alginic acid). “Static MC” refers to control group 1. “Agitation MC” refers to control group 2. The left and center are microscope images in the bright field, and the right is a fluorescence microscope image.

From FIG. 9B, the cell proliferation rate in the cell structure (containing gel-like alginic acid) was remarkably high. On the other hand, in the control group 2, the cell proliferation rate was significantly reduced.

In particular, the control group 2 is a conventional suspension culture method using a microcarrier, and it has been said that inefficient proliferation, cell death, and change in properties are problems. Further, on the 4th day of culturing, although the agglomerates are small, the agglutination becomes too large as in the control group 1 by continuing the culturing.

On the other hand, in the cell structure (containing gel-like alginic acid), the influence of shear stress was reduced because it was a static culture, and the generation of dead cells was suppressed by controlling the size of the agglomerates. It was inferred.

From the above, it was suggested that cells can be cultured with a high cell viability and a high cell proliferation rate in the cell structure (gel-like alginate-free). In addition, the cell structure (containing gel-like alginic acid) has a low cell proliferation rate and a high cell survival rate, suggesting that it can be used for storing or transporting cells.

According to the three-dimensional cell culture method of the present embodiment, cell death due to excessive aggregation of microcarriers and cells can be suppressed. According to the cell structure of the present embodiment and the method for producing the same, the size of microcarriers and cell aggregates can be controlled, and the cell viability and cell proliferation rate can be improved. According to the cell transport method of the present embodiment, cells can be safely and easily transported while maintaining the cell viability.

1a ... a first solution containing a polysaccharide, 1b ... a gel-like polysaccharide, 2 ... a microcarrier, 3 ... an adhesive cell, 4 ... a second solution containing a coagulant for the polysaccharide, 5 ... a polyamino acid. Third solution containing, 6 ... polyamino acid, 7 ... fourth solution containing a solubilizer for gel-like polysaccharide, 8 ... spinner flask, 9 ... medium, 10 ... cell structure (containing gel-like polysaccharide) ), 20 ... Cellular structure (without gel-like polysaccharide)

Claims (13)

The first solution containing the polysaccharide, the microcarriers, and the adhesive cells are mixed, and a second solution containing a coagulant for the polysaccharide is added to gel the polysaccharide to obtain the above-mentioned A gelation step of obtaining a fibrous mixture containing the microcarriers, the adhesive cells and the gel-like polysaccharide .
A first coating step of adding a third solution containing the polyamino acid to coat the fibrous mixture containing the microcarriers, the adhesive cells and the gel-like polysaccharide with the polyamino acid.
A removal step of adding a fourth solution containing the gel-like polysaccharide solubilizer to remove the polysaccharide, and a removal step.
A culture step of culturing a fibrous mixture containing the microcarriers and the adhesive cells coated with the polyamino acid, and a culture step.
A three-dimensional culture method for cells comprising. After the first coating step and before the removal step, the first solution and the second solution are further added to the microcarriers coated with the polysaccharide, the adhesiveness. The three-dimensional culture method for cells according to claim 1, further comprising a second coating step of coating the cell and the fiber-like mixture containing the gel-like polysaccharide with the gel-like polysaccharide. The method for three-dimensional culturing cells according to claim 1 or 2, wherein the first solution further comprises an extracellular matrix. Contains microcarriers and adhesive cells
The mixture containing the microcarriers and the adhesive cells is coated with polyamino acids .
A cellular structure that is fibrous . The cell structure according to claim 4, wherein the mixture further contains a gel-like polysaccharide. The cell structure according to claim 4 or 5, wherein the mixture further comprises an extracellular matrix. The cell structure according to any one of claims 4 to 6, wherein the mixture coated with the polyamino acid is further coated with a gel-like polysaccharide. The microcarrier, the adhesive cell, and the first solution containing the polysaccharide are mixed, a second solution containing a coagulant for the polysaccharide is added, and the polysaccharide is gelled to obtain the above-mentioned A gelation step of obtaining a fibrous mixture containing the microcarriers, the adhesive cells and the gel-like polysaccharide .
A first coating step of adding a third solution containing a polyamino acid to coat a fibrous mixture containing the microcarriers, the adhesive cells and the gel-like polysaccharide with the polyamino acid.
A method for producing a cell structure comprising. The cell structure according to claim 8, further comprising a removal step of adding a fourth solution containing the solubilizing agent for the gel-like polysaccharide after the first coating step to remove the polysaccharide. Manufacturing method. After the first coating step and before the removal step, the first solution and the second solution are further added to the microcarriers coated with the polysaccharide, the adhesiveness. The method for producing a cell structure according to claim 9, further comprising a second coating step of coating the fiber-like mixture containing the cells and the gel-like polysaccharide with the gel-like polysaccharide. The method for producing a cell structure according to any one of claims 8 to 10, wherein the first solution further contains an extracellular matrix. A method for transporting cells using the cell structure according to any one of claims 5 to 7. A method for preserving cells, comprising a preservation step of preserving the cell structure obtained by the production method according to claim 8 at 4 ° C. or higher and 40 ° C. or lower.

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