JP6558432B2 - Tissue preparation method - Google Patents

Description

The present invention relates to a tissue production method. In particular, the present invention relates to a method suitable for manufacturing a sheet-like cell tissue body in which a plurality of cells are laminated in the thickness direction.

A living tissue produced by culturing cells on a culture carrier such as collagen is not suitable as a tissue for transplantation for medical purposes because of its low cell density. A technique for producing a tissue such as a cell sheet having a high cell density suitable for transplantation is important in the field of tissue engineering. However, there are some problems in the conventional method for producing a high-density tissue.

It is generally widely practiced to seed cells on a culture support to form a cell sheet.
For the purpose of facilitating cell sheet detachment, a technology has been developed to promote cell detachment by providing a temperature-responsive polymer layer on the cell adhesion surface, and it is possible to collect cell sheets that contain almost no foreign matter. . However, the cell sheet formed by this method is usually composed of a single layer or three or less cell layers, and in order to achieve multilayering, it is necessary to superimpose a plurality of cell sheets. Since cell sheets are extremely thin and difficult to handle, it is not easy to superimpose a plurality of cell sheets, which is troublesome.

On the other hand, a method of applying centrifugal force to cells in cell culture has been proposed. For example, in Patent Document 1, a step of seeding cells on a substrate, a step of pressurizing the cells against the substrate by centrifugal force, and culturing the cells to form a sheet-like cell culture. Disclosed is a method for producing a released sheet-shaped cell culture, which comprises a step and a step of releasing the formed culture from a substrate.

In addition, in the Patent Document 2, the present inventors added cells and extracellular matrix components to a culture vessel having an inner bottom surface that is non-cell-adhesive or can be changed to cell-non-adhesive. A cell addition step of adding the culture medium contained, and a cell culture under a condition in which cell-cell adhesion is formed while acting a centrifugal force toward the inner bottom surface on the cells added to the culture vessel, and the cells are bonded A tissue culturing method is proposed, which includes a cell culturing step for forming a tissue and a detaching step for detaching and collecting the tissue obtained in the cell culturing step from the inner bottom surface. In this technology, by adding a component prepared from cells derived from the same individual as the cultured cells as an extracellular matrix component, all biological components of the finally produced tissue are derived from the same individual. This has the advantage that the safety in transplanting the tissue is enhanced and the cell-cell adhesion is accelerated as compared with the case where the extracellular matrix is secreted from the cultured cells and the cells are adhered to each other.

JP 2010-226962 A (Claims 1 and 4) JP 2010-161952 A (Claims 1, 3 and 4)

However, in the technique of the above (Patent Document 2), even when an extracellular matrix is separately added, it may take several hours to obtain a sheet-like cell tissue body, thereby reducing damage to cells. From the viewpoint, it has been desired to produce a tissue in a shorter time. In recent years, in particular, it has been reported that differentiation may be initiated by applying mechanical stimulation to, for example, induced pluripotent stem cells (iPS cells). Therefore, an undifferentiated state is maintained by efficient tissue preparation. It was hoped to do.

In view of the above-described conventional situation, an object of the present invention is to provide a method capable of producing a tissue more efficiently using centrifugal force. Another object of the present invention is to provide a sheet-like cell tissue obtained by the method.

As a result of intensive studies by the present inventors, it has been found that the above-mentioned problems can be solved by adding cell-adhesive fine particles together with cells to a culture solution, and the present invention has been completed. That is,
The gist of the present invention is as follows.

(1) a cell addition step of adding a culture solution containing cells and cell-adhesive microparticles to a culture vessel having an inner bottom surface that is non-cell-adhesive or capable of changing to cell-non-adhesion;
A cell culturing step of performing cell culture while acting a centrifugal force in the direction of the inner bottom surface on the cells applied to the culture vessel, and forming a tissue by adhering the cells;
And a peeling step of peeling and recovering the obtained tissue from the inner bottom surface.
(2) The method for producing a tissue according to (1), wherein an average particle diameter of the cell adhesive fine particles is 500 nm to 10 μm.
(3) The amount of the cell-adhesive fine particles added to the culture container is 0.00 per cell.
The method for producing a tissue according to (1) or (2) above, which is 02 ng to 20 ng.
(4) The above-mentioned (1) to (1), wherein the cell adhesive fine particles are gelled collagen fine particles.
The method for producing a tissue according to any one of 3).
(5) A sheet-like cell tissue produced by the method according to any one of (1) to (4) above.
(6) A cell culture kit used in the method according to any one of (1) to (4) above,
A culture vessel having an inner bottom surface that is non-cell-adherent or capable of changing to cell-non-adherent;
The cell culture kit, comprising cell adhesive fine particles.

According to the present invention, a biological tissue having a high cell density, such as a sheet-like cell tissue body, can be produced more efficiently. The produced tissue is suitably used for therapeutic purposes such as transplantation.

It is a figure for demonstrating each process of the preparation method of the structure | tissue which concerns on this invention. 2 is a photograph showing a sheet-like cell tissue produced in Example 1. FIG. 2 is an enlarged micrograph of a sheet-like cell tissue produced in Example 1.

Hereinafter, the present invention will be described in detail.
1. Cell addition step In the cell addition step, at least the inner bottom surface of the inner surface of the container part is non-cell-adherent,
In this step, a culture solution containing cells and cell-adhesive microparticles is added to a culture vessel that can be changed to non-cell-adhesive.

The culture container used in the present invention is not limited as long as at least the inner bottom surface of the inner surface of the container part that stores the culture solution is non-cell-adhesive or can be changed to cell-non-adhesive. In particular,
Of the inner surface of the container part containing the culture solution, all the surfaces that come into contact with the cells (for example, the inner bottom surface and the inner surface of the container) are non-cell-adhesive or can be changed to cell-non-adhesive. Preferably there is.

In the present invention, the non-cell-adhesive surface is a hydrophilic surface, specifically 20 ° C.
And a surface having a static water contact angle of 45 ° or less. Such a surface can be obtained, for example, by forming a film of an organic compound having a carbon-oxygen bond on the surface of the substrate. Or you may comprise the base material itself with the material which has hydrophilic property.

The material of the base material for forming the hydrophilic film on the surface is not particularly limited, and specifically, inorganic materials such as metal, glass, ceramic, silicon, elastomer, plastic (for example,
Polyester resin, polyethylene resin, polystyrene resin, polypropylene resin, ABS
Resin, polyamide resin, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin, and the like.

The non-cell-adhesive surface can be formed by a hydrophilic film formed of an organic compound having a carbon-oxygen bond and having a static water contact angle of 45 ° or less. Here, the carbon-oxygen bond means a bond formed between carbon and oxygen, and is not limited to a single bond but may be a double bond. Examples of the carbon oxygen bond include a C—O bond, a C (═O) —O bond, and a C═O bond.

Examples of main raw materials for the hydrophilic coating include hydrophilic organic compounds such as water-soluble polymers, water-soluble oligomers, water-soluble low-molecular compounds, surface active substances, and amphiphilic substances. These are optionally physically or chemically cross-linked with each other and physically or chemically bonded to the substrate to form a hydrophilic coating.

Specific water-soluble polymers include polyalkylene glycol and derivatives thereof, polyacrylic acid and derivatives thereof, polymethacrylic acid and derivatives thereof, polyacrylamide and derivatives thereof, polyvinyl alcohol and derivatives thereof, zwitterionic polymers, A polysaccharide etc. can be mentioned. Examples of the molecular shape include linear, branched, and dendrimers. More specifically, polyethylene glycol, a copolymer of polyethylene glycol and polypropylene glycol, such as Pluronic (registered trademark) F108, Pluroni
c (registered trademark) F127, poly (N-isopropylacrylamide), poly (N-vinyl-2-pyrrolidone), poly (2-hydroxyethyl methacrylate), poly (methacryloyloxyethylphosphorylcholine), methacryloyloxyethylphosphoryl Examples include, but are not limited to, a copolymer of Lucolin and an acrylic monomer, dextran, and heparin.

Specific water-soluble oligomers and water-soluble low-molecular compounds include alkylene glycol oligomers and derivatives thereof, acrylic acid oligomers and derivatives thereof, methacrylic acid oligomers and derivatives thereof, acrylamide oligomers and derivatives thereof, and saponified vinyl acetate oligomers and the like Derivatives, oligomers consisting of zwitterionic monomers and their derivatives, acrylic acid and its derivatives, methacrylic acid and its derivatives, acrylamide and its derivatives, zwitterionic compounds, water-soluble silane coupling agents, water-soluble thiol compounds, etc. Can do. More specifically, ethylene glycol oligomer, (N-isopropylacrylamide) oligomer, methacryloyloxyethylphosphorylcholine oligomer, low molecular weight dextran, low molecular weight heparin, oligoethylene glycol thiol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene Examples include, but are not limited to, glycol, 2- [methoxy (polyethyleneoxy) -propyl] trimethoxysilane, and triethylene glycol-terminated-thiol.

The average thickness of the hydrophilic coating is preferably 0.8 nm to 500 μm, and 0.8 nm to 100 μm.
m is more preferable, 1 nm to 10 μm is further preferable, and 1.5 nm to 1 μm is most preferable. An average thickness of 0.8 nm or more is preferable because it is not easily affected by a region not covered with the hydrophilic film on the substrate surface. Moreover, if the average thickness is 500 μm or less, coating is relatively easy.

As a method of forming a hydrophilic film on the substrate surface, a method of directly adsorbing a hydrophilic organic compound to a substrate, a method of directly coating a hydrophilic organic compound on a substrate, or a coating of a hydrophilic organic compound on a substrate A method of performing a crosslinking treatment later, a method of forming a hydrophilic film in a multi-stage manner in order to increase the adhesion to the substrate, a base layer is formed on the substrate in order to increase the adhesion with the substrate,
Next, a method of coating a hydrophilic organic compound, a method of forming a polymerization initiation point on the substrate surface, and then polymerizing a hydrophilic polymer brush can be exemplified.

Among the above-mentioned film forming methods, particularly preferable methods include a method of forming a hydrophilic film in a multistage manner, and a base layer is formed on the substrate in order to improve adhesion to the substrate, and then hydrophilic A method of coating an organic compound can be mentioned. With these methods,
This is because it is easy to improve the adhesion of the hydrophilic organic compound to the substrate. Less than,"
This will be described using the term “bonding layer”. The binding layer means a layer existing between the outermost hydrophilic coating layer and the base material in the case of forming a hydrophilic coating in a multistage manner, and a base layer is provided on the base material surface. When a hydrophilic organic compound is coated on the surface, it means the underlayer. The binding layer is preferably a layer containing a material having a binding portion (linker). Examples of combinations of the linker and the functional group at the end of the material to be bonded to the linker include epoxy group and hydroxyl group, phthalic anhydride and hydroxyl group, carboxyl group and N-hydroxysuccinimide, carboxyl group and carbodiimide, amino group and glutaraldehyde, etc. Is mentioned. In each combination, any of them may be a functional group on the linker side. In these methods, a bonding layer made of a material having a linker is formed on a substrate before coating with a hydrophilic organic compound. The density of the material in the tie layer is an important factor that defines the bond strength. The density can be easily evaluated using the contact angle of water on the surface of the bonding layer as an index. For example, in the case of a silane coupling agent having an epoxy group at the end (epoxy silane), the water contact angle of the substrate surface to which the epoxy silane has been added is typically 45 ° or more, preferably 47 ° or more. Then, a surface having sufficient cell non-adhesiveness can be obtained by adding an ethylene glycol-based material or the like in the presence of an acid catalyst.

The cell contact region on the inner surface including the inner bottom surface of the culture vessel used in the present invention is preferably cell non-adhesive from the viewpoint of ease of tissue detachment. However, it may be a surface that can be changed to cell non-adhesiveness upon peeling. Such a surface is formed by immobilizing at least one stimulus-responsive polymer selected from the group consisting of a temperature-responsive polymer, a pH-responsive polymer, and an ion-responsive polymer on the surface of the substrate. be able to. The stimulus-responsive polymer is particularly preferably a temperature-responsive polymer, but is not limited thereto.

The temperature-responsive polymer that can be suitably used in the present invention is hydrophobic at the cell culture temperature (usually about 37 ° C.) and hydrophilic at the temperature at which the cultured cell tissue is recovered. . The temperature at which the temperature-responsive polymer changes from hydrophobic to hydrophilic (the critical dissolution temperature (T) in water) is not particularly limited, but from the viewpoint of ease of recovery of the cell tissue after culturing. The temperature is preferably lower than the cell culture temperature. By including such a temperature-responsive polymer component, since cell scaffolds (cell adhesion surfaces) are sufficiently secured during cell culture, cell culture can be performed efficiently. On the other hand, at the time of collection of the cell tissue after culturing, the hydrophobic part is changed to hydrophilic, and the cultured cell tissue is separated from the cell culture substrate, thereby further collecting the cell tissue. Can be easily.
In particular, a temperature-responsive polymer that exhibits hydrophilicity at a temperature lower than a predetermined critical dissolution temperature and exhibits hydrophobicity at a temperature equal to or higher than the same temperature is preferable. The critical solution temperature in such a temperature-responsive polymer is particularly called the lower critical solution temperature.

Specifically, the temperature-responsive polymer that can be suitably used in the present invention is preferably a polymer having a lower critical solution temperature T of 0 ° C. to 80 ° C., preferably 0 ° C. to 50 ° C. It is not preferable that T exceeds 80 ° C. because cells may die. If T is lower than 0 ° C., the cell growth rate is generally extremely reduced or the cells are killed. Such suitable polymers include acrylic polymers or methacrylic polymers.
As a suitable polymer, specifically, poly-N-isopropylacrylamide (T = 3
2 ° C), poly-Nn-propylacrylamide (T = 21 ° C), poly-Nn-propylmethacrylamide (T = 32 ° C), poly-N-ethoxyethylacrylamide (T = about 35 ° C), Poly-N-tetrahydrofurfurylacrylamide (T = about 28 ° C.), poly-
Examples thereof include N-tetrahydrofurfuryl methacrylamide (T = about 35 ° C.) and poly-N, N-diethylacrylamide (T = 32 ° C.). Other polymers include, for example, poly-N-ethylacrylamide, poly-N-isopropylmethacrylamide, poly-
Alkyl-substituted cellulose derivatives such as N-cyclopropylacrylamide, poly-N-cyclopropylmethacrylamide, poly-N-acryloylpyrrolidine, poly-N-acryloylpiperidine, polymethyl vinyl ether, methylcellulose, ethylcellulose, hydroxypropylcellulose, and polypolypropylene A polyalkylene oxide block copolymer represented by a block copolymer of oxide and polyethylene oxide,
Examples include polyalkylene oxide block copolymers.

Examples of the monomer for forming these polymers include a monomer having a monomer homopolymer having T = 0 ° C. to 80 ° C. and capable of being polymerized by irradiation. Examples of the monomer include (meth) acrylamide compounds, N-
(Or N, N-di) alkyl-substituted (meth) acrylamide derivative having a cyclic group (
A meth) acrylamide derivative, a vinyl ether derivative, etc. are mentioned, 1 or more types of these can be used. When one type of monomer is used alone, the polymer formed on the substrate is a homopolymer, and when multiple types of monomers are used together, the polymer formed on the substrate is a copolymer. Both forms are encompassed by the present invention. In addition, when it is necessary to adjust T depending on the type of proliferating cells, other monomers other than those described above may be further added for copolymerization. Furthermore, a graft or block copolymer of the above polymer and other polymer used in the present invention, or a mixture of the above polymer and another polymer may be used. Moreover, it is also possible to crosslink within a range where the original properties of the polymer are not impaired.

As the pH responsive polymer and the ion responsive polymer, those suitable for the cell tissue to be prepared can be appropriately selected.

The cells used in the present invention are not particularly limited as long as they are adherent cells. Examples of such cells include liver cells that are liver parenchymal cells, Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, Epidermal cells such as epidermal keratinocytes, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells such as corneal epithelial cells, mammary cells, pericytes, muscle cells such as smooth muscle cells and cardiomyocytes, kidneys Examples thereof include cells, pancreatic islets of Langerhans, nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, bone cells and the like. These cells may be primary cells collected directly from tissues or organs, or may be obtained by substituting them for several generations. Furthermore, these cells are undifferentiated embryonic hepatocytes, pluripotent stem cells such as pluripotent mesenchymal stem cells, unipotent stem cells such as vascular endothelial progenitor cells having unipotency, and differentiation is completed. Any of the obtained cells may be used. The cells may be cultivated as a single species, or two or more cells may be co-cultured.

These cells are cultured in advance by a conventional method, the culture is treated with trypsin treatment or the like, and is stored in a culture vessel in a state of being suspended in a culture solution. As a culture solution, any cell culture medium usually used in this technical field can be used without particular limitation. For example, depending on the type of cells used, MEM medium, BME medium, DME medium, αMEM medium, IMDM
Medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium and RP
MI1640 medium, etc., published by Asakura Shoten "Tissue Culture Technology 3rd Edition" edited by Japanese Society for Tissue Culture 58
A basal medium as described on page 1 can be used. In addition, serum (
Fetal bovine serum, etc.), various growth factors, antibiotics, amino acids and the like may be added. Gib
A commercially available serum-free medium such as co-serum-free medium (Invitrogen) can be used.
Considering the clinical application of the finally obtained cell tissue, it is preferable to use a medium that does not contain animal-derived components.

The present invention is characterized in that the culture solution contains cell-adhesive fine particles in addition to the cells. Cell-adhesive microparticles are microparticles having a function of being precipitated together with cells by centrifugal force and adhering between cells to form a tissue. Preferably, when a centrifugal force of 1440 G is applied, it is desirable to precipitate on the inner bottom surface of the culture vessel within a normal cell culture time (less than 30 minutes to several hours). Examples include aggregates of extracellular matrix molecules such as fibrin, fine particles composed of cell adhesive molecules such as gelatin, and fine particles composed of organic or inorganic materials whose surfaces are coated or modified with these cell adhesive molecules. For example, fine particles obtained by gelling various proteins or polysaccharides are preferably used.

Specific examples of suitable cell adhesive fine particles include gelled collagen fine particles and gelatin fine particles. Collagen is a rod-shaped basic protein with a length of about 300 nm and a thickness of about 1.5 nm, but even if this collagen is added to the culture solution as it is, it may not be sufficient from the viewpoint of more efficient tissue preparation. is there. The inventors of the present invention have found that cells with high cell density can be efficiently obtained by gelling collagen and finely pulverizing the resulting collagen gel to firmly adhere cells to each other. In order to produce such collagen gel fine particles, for example, collagen, which is a basic protein, is first solubilized under acidic conditions to prepare a collagen solution, and this collagen solution is subjected to physiological conditions (neutral, temperature 37). A large number of collagen molecules are associated and gelled. When collagen is sufficiently gelled, or when a gelation degree of preferably 5% to 95% is obtained, shearing force is applied to the collagen gel by pipetting, ultrasonic treatment, etc., and the gel is pulverized into fine particles. Can be obtained.

If the size of the cell adhesive fine particles is too small, it is difficult to precipitate due to centrifugal force. Conversely, if the size is too large, the cell density of the tissue to be produced decreases. It is preferably sufficiently smaller than the cells to be cultured. Specifically, the average particle diameter is 500 nm to 10 μm, particularly 1 μm to 3 μm, although it varies depending on the type of cells. The average particle size of the cell-adhesive fine particles is within a desired range by appropriately changing the pulverization conditions (such as ultrasonic treatment time) when the gel is pulverized into fine particles as described above, for example. Can be controlled. Alternatively, the average particle diameter may be adjusted by sieving after making fine particles. Here, the average particle diameter is an average particle diameter by microscopic observation. The average particle diameter obtained by microscopic observation is obtained, for example, by performing microscopic observation, measuring the particle diameters of 100 arbitrary fine particles with image processing software and the like, and averaging the number. The particle diameter refers to the average value of the major axis diameter and minor axis diameter of the fine particles. In addition, the specific gravity of the cell-adhesive fine particles is set such that when a centrifugal force is applied, the cell-adhesive fine particles are uniformly located between the cells and the fine particles are not biased as much as possible in the produced tissue. Specifically, although it varies depending on the type of cell, it is within ± 20% of the specific gravity of the cell, preferably within ± 10%.

The amount of the cell-adhesive fine particles added to the culture vessel is preferably smaller if the tissue can be formed because the cell density in the tissue becomes higher. Specifically, although depending on the average particle size of the cell adhesive fine particles, the amount of the cell adhesive fine particles is 0.002 ng to 2 per cell.
00 ng is preferable, and 0.02 ng to 20 ng is particularly preferable. As an example, when a tissue composed of mouse fibroblasts is prepared, a suitable amount of collagen gel microparticles having an average particle size of about 3 μm per 1 × 10 ⁷ cells is 0.3 mg (0.03 ng per cell). Degree.

2. Cell Culture Process The cell culture process will be described with reference to FIG. In the cell culturing step, cell culture is performed while applying centrifugal force G in the direction of the inner bottom surface 1a to the culture solution 4 containing the cells 2 and the cell-adhesive fine particles 3 contained in the culture vessel 1, and the cells 2 This is a step of forming a tissue by bonding the gaps. FIG.
A and B correspond to this step. In this step, the cells 2 are brought into close contact with the inner bottom surface 1a corresponding to the shape of the inner bottom surface 1a by the centrifugal force G, and the cells 2 adhere to each other through the cell adhesive fine particles 3 in this state, and the tissue having a desired shape is obtained. Is formed.

The magnitude of the centrifugal force G can be appropriately selected as long as the tissue can be formed without adversely affecting the function of the cell 2. For example, 2G-1440G is preferable and 2G-720G is more preferable. A culture vessel 1 containing a culture solution 4 containing cells 2 and cell adhesion fine particles 3
Is placed in a centrifuge and centrifugal force G can be applied by performing a centrifugal operation.

Cell culture is performed under conditions where cell-cell adhesion is formed. Here, “the conditions under which cell-cell adhesion is formed” refers to conditions under which cells can act to allow cells to adhere to each other. Although it varies depending on the type of cells to be cultured, for example, the temperature condition is preferably 20 ° C. to 40 ° C. The atmospheric gas condition is preferably a carbon dioxide concentration of 3% to 5%. 5-24 hours are preferred. In the present invention, by containing the cell adhesion fine particles 3 having a relatively large particle size,
The adhesion between cells is further accelerated, and the tissue can be obtained efficiently.

When culturing cells, leave the culture vessel in an incubator filled with the desired atmospheric gas (for example, carbon dioxide concentration 5%) for several minutes, and then cover the culture vessel so that the atmospheric gas does not change. Weighted culture can be performed. Or it is not limited to this, For example, if it is in the incubator where atmosphere gas and temperature were controlled, weight culture can also be performed in the state where the culture vessel was opened.

It is sufficient that cell-cell adhesion is formed in the cell culture process, and it is not essential to increase the number of cells by proliferation. By appropriately adjusting the number of cells in the culture solution, the thickness of the tissue to be produced (that is, the number of cells stacked in the thickness direction of the tissue) can be controlled. Since it is not necessary to proliferate cells in the cell culture process, a tissue can be obtained in a relatively short time.
In addition, the thickness and shape of the tissue can be freely adjusted.

Moreover, according to the method of the present invention, it is possible to obtain a tissue in which cells are densified. A tissue in which cells are densified is preferably used for transplantation.

3. Peeling process A peeling process is a process which peels and collect | recovers the obtained structure | tissue from the inner bottom face 1a of the culture container 1 after completion | finish of centrifugation operation. C in FIG. 1 corresponds to this step. For example, the cells can be detached by a physical operation such as a pipetting operation. If the inner bottom surface 1a of the culture vessel 1 is a non-cell-adhesive surface, this operation is easy, and the tissue can be produced and peeled and collected in a short time. As shown in FIG. 1, a sheet-like cell tissue body 5 as a tissue can be obtained along the flat shape of the inner bottom surface 1a. When the inner bottom surface 1a of the culture vessel 1 is a surface that changes to non-cell-adhesive properties, such as a stimulus-responsive polymer, an environment in which the cell is non-adhesive (for example, a temperature below the lower critical temperature) The peeling operation is performed at.

As an example of a method of peeling while maintaining the shape of the tissue, first, gelatin is flowed on the surface of the tissue formed on the inner bottom surface of the cell non-adhesive to gel, and further a holding substrate such as a PET film Thus, the gelatin gel can be retained, and then the tissue can be peeled off and collected together with the gelatin gel from the inner non-adhesive bottom surface. The tissue thus peeled and collected is further
It can be transplanted to a desired subject by bringing it into close contact with the transplant subject and dissolving gelatin at 37 ° C.

The present invention also provides a cell culture kit comprising a culture vessel having an inner bottom surface that is non-cell-adhesive or capable of changing to cell-non-adhesion, and the cell-adhesive microparticles described above. It is. By performing cell culture as described above using this kit, a desired tissue such as a sheet-like cell tissue body can be efficiently obtained.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, it is not limited to this.

(Example 1)
The whole inner bottom surface is made non-adherent by chemically applying polyethylene glycol to the entire inner bottom surface of a glass container having a bottom diameter of 2 cm, and suspended in DMEM medium containing 10% fetal bovine serum. Mouse fibroblasts 4 × 10 ⁶ and fine particles of atelocollagen (low antigenic collagen) gel (average particle size 3 μm, the amount of fine particles per cell was 0.03)
ng) was inoculated in a culture vessel, and cultured for 0.5 hour in an environment of 37 ° C. and 5% CO ₂ concentration while applying a centrifugal force of 720 G in the direction of the inner bottom surface with a centrifuge. After culturing, the centrifuge was stopped, and physical force by water flow was applied to the inner bottom surface, so that the sheet-like cellular tissue body could be easily and quickly peeled and collected from the inner bottom surface without destroying the tissue structure (Fig. 2).

When the collected sheet-like cell tissue was observed with a microscope, it was confirmed that the cells were in close contact with each other and a tissue with a high cell density was formed (FIG. 3). In addition, in order to examine the presence or absence of cell survival in the obtained tissue, the cells were dispersed by treating the sheet-like cell tissue with trypsin and EDTA, and then a cell viability assay kit (product name: cell double staining kit) , Manufacturer: Dojindo, product number: CS01), the cell viability before applying the centrifugal force was 90%, whereas the cells of the obtained sheet-like cell tissue were The survival rate was 90%. From this result, it was clarified that cells hardly die by the method of the present invention.

(Comparative Example 1)
Cell culture was performed in the same manner as in Example 1 except that atelocollagen gel fine particles were not contained in the culture solution. After culturing, when the centrifuge was stopped and a physical force by water flow was applied to the inner bottom surface, the tissue structure collapsed and a sheet-like cell tissue body could not be obtained.

DESCRIPTION OF SYMBOLS 1 Culture container 1a Inner bottom face 2 Cell 3 Cell adhesion microparticle 4 Culture solution 5 Sheet-like cell organization G Centrifugal force

Claims (4)

A sheet-like cell tissue containing cells and cell-adhesive microparticles made of gelled protein or polysaccharide ,
The sheet-like cell tissue body, wherein the cell-adhesive fine particles are disposed between the cells along at least the in-plane direction of the sheet-like cell tissue body.   The sheet-like cell tissue body according to claim 1, wherein the cell-adhesive fine particles have an average particle size of 500 nm to 10 μm.   The sheet-like cell tissue body according to claim 1 or 2, wherein the amount of the cell adhesive fine particles is 0.02 ng to 20 ng per cell.   The sheet-like cell tissue according to any one of claims 1 to 3, wherein the cell adhesive fine particles are gelled collagen fine particles.

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