Method for producing cell sheet containing hyaluronic acid and polyethylene glycol

Abstract

The present invention relates to a method for producing a cell sheet composed of cells only without a support and configured to have multiple layers without a separate lamination step, and to a cell sheet produced by the method.

Description

[Technical field]

[0001] The present invention relates to a method for constructing a cell sheet in a non-adherent state without a support and for producing a cell sheet constructed in multiple layers without a separate lamination step, and to a cell sheet produced by the method. [Background technology]

[0002] Since cells exhibit higher activity when they maintain an attached state, in order to maximize the activity of transplanted cells, cell sheets or scaffold-based cell complexes that maintain cell-cell or cell-substrate bonds are more advantageous than suspensions collected in the form of single cells.

[0003] For the reasons mentioned above, various techniques have been developed to form transplantable cell sheets, and cells cultured in this sheet state have the advantage that cell activity can be maintained at a high level by maintaining cell-cell contact and pericellular matrix molecules.

[0004] However, cell structures cultured in a single-layer sheet state have the disadvantage that they can easily become structurally damaged during handling for cell transplantation, etc., and there are also problems such as the need for additional technology to create multi-layered cell sheets with high integrity to ensure the thickness of the regenerated tissue.

[0005] To date, cell therapy in the form of injection or transplantation of cells themselves has been the mainstream, but due to the increasing demand for tissue engineering preparations, which are produced by culturing transplantable tissues outside the body, there is a need for continuous development in related fields. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method for producing a slab-shaped cell slap, which includes a processing step of hyaluronic acid (HA) and polyethylene glycol (PEG).

[0007] Another object of the present invention is to provide a plate-shaped cell slap produced by the above-mentioned production method.

[0008] It is still another object of the present invention to provide a graft material for cartilage repair, comprising the above-mentioned cell slap and a biodegradable polymeric substance. [Means for solving the problem]

[0009] In order to achieve the above-mentioned objective, one aspect of the present invention provides a method for producing a cell sheet, comprising: a) the steps of precipitating and culturing cells in a growth medium containing hyaluronic acid; and b) adding a growth medium containing polyethylene glycol.

[0010] Specifically, the cell sheet produced by the above-mentioned production method may be in the form of a plate.

[0011] In the present invention, the term "cell sheet" refers to one or more cells arranged in a layer, and the morphology may be, but is not limited to, a plate-like shape. The term "multi-layered cell sheet" refers to an assembly of cell sheets formed by stacking one or more of the cell sheets vertically. Hereinafter, the term "cell sheet" described in this specification is used to refer to both a cell sheet having a single layer and a multi-layered cell sheet.

[0012] In the present invention, the term "lamination" or "formation" means that thin film layers are stacked in the order in which they are produced. There are no particular limitations on the laminated structure, so long as it is produced by a method known in the art.

[0013] In the present invention, the "hyaluronic acid (HA)" is a complex polysaccharide consisting of amino acids and uronic acid, and is a polymer compound consisting of N-acetylglucosamine and glucuronic acid. It is mainly present in the vitreous body of the eye and the umbilical cord, and plays a role in preventing the invasion of bacteria and the penetration of toxic substances. In addition, hyaluronic acid is a drug used for purposes such as auxiliary agents in various ophthalmic surgeries, intra-articular injections, artificial tears, and wound healing, and is a type of polysaccharide that can contain water equivalent to 300 to 1000 times its own weight and is known to have excellent moisturizing properties. However, there has been no report on a method of using hyaluronic acid together with PEG to produce a cell sheet as in the present invention.

[0014] In the present invention, the "polyethylene glycol (PEG)" is a type of surfactant that stably maintains products during the production of cosmetics and acts like glycerin, and is a chemical ingredient that is used in a variety of applications such as lotions, creams, and shampoos. However, there has been no report of a method in which PEG is used together with HA to prepare a cell sheet as in the present invention.

[0015] Specifically, the cells used in the cell sheet may be one or more selected from the group consisting of epidermal cells, fibroblasts, hepatocytes, mesodermal stem cells, and chondrocytes, and more specifically, may be chondrocytes, but are not limited thereto.

[0016] In one embodiment of the present invention, the chondrocytes are derived from the meniscus of a rabbit, and a cell slap is prepared using the chondrocytes, but the present invention is not limited thereto.

[0017] The cells used in the cell sheet may be cells differentiated from human pluripotent stem cells.

[0018] In the present invention, the term "human pluripotent stem cells (PSCs)" refers to stem cells that have the ability to differentiate into any of the three germ layers that constitute the human body, i.e., endoderm, mesoderm, and ectoderm. The human pluripotent stem cells are considered to be very useful materials for clinical trials for the treatment of various diseases, development of new drugs, toxicity evaluation, disease modeling, and research on early embryonic development.

[0019] Specifically, the cell sheet can be characterized in that cell aggregation is suppressed.

[0020] In one embodiment of the present invention, the cell sheet was formed without a separate scaffold in a non-adherent state. It was confirmed that the cell sheet thus formed significantly suppressed cell aggregation, hardly produced spheroids, and the cells maintained their sheet shape by remaining settled to the bottom in the culture medium (Experimental Example 1, Figures 2 to 4).

[0021] This result is believed to be due to the differential effects of hyaluronic acid (HA) and polyethylene glycol (PEG) present in the culture medium, which regulates cell behavior and changes the tendency for spheroid formation through cell aggregation. Hyaluronic acid (HA), which has hydrogel properties, distributes the medium components evenly and maintains intercellular bonds, while polyethylene glycol (PEG) in the medium exerts a repulsive force on the cells, which generates a force that tries to minimize the interface between the PEG in the medium and the cells, maintaining the shape of a plate-like cell sheet (cell slap).

[0022] In the present invention, the term "hydrogel" refers to a material or form that can contain a large amount of water and can easily transmit and transfer substances necessary for cell survival, such as oxygen, water, water-soluble nutrients, enzymes, and polypeptides such as cytokines, waste products, etc., and is generally biocompatible. The shape or form of the hydrogel is not particularly limited as long as it can be integrated into the cell sheet, and various shapes or forms such as fine particles, granules, films, tubes, disks, nets, meshes, porous, suspensions, or dispersions may be used. Among them, hydrogel particles obtained by solidifying an aqueous solution containing colloidal particles are preferred. Particles made of any material can be used as long as they have the properties of the hydrogel. For example, water-soluble, hydrophilic, or water-absorbent synthetic polymers such as polyacrylamide, polyacrylic acid, polyhydroxyethyl methacrylate, polyvinyl alcohol, polylactic acid, polyglycolic acid, and polyethylene glycol; and hydrogel particles made of chemically crosslinked polysaccharides, proteins, and nucleic acids. Examples of polysaccharides include, but are not limited to, glycosaminoglycans such as hyaluronic acid and chondroitin sulfate, starch, glycogen, agarose, pectin, and cellulose.Examples of proteins include, but are not limited to, collagen and its hydrolyzate, gelatin, proteoglycan, fibronectin, vitronectin, laminin, entactin, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, etc. Specifically, particles made of a material that is biocompatible and decomposed by cells in the body are suitable for the present invention, and most specifically, they may be hyaluronic acid and polyethylene glycol.

[0023] Specifically, the cell sheet may be composed of multiple layers of cells, and each cell layer constituting the multiple layers may be characterized as having structural continuity while maintaining integrity between the cell layers.

[0024] Existing cell sheets are usually formed using a scaffold as a base, but such cell sheets formed using a scaffold as a base have problems in that the cells are not uniformly distributed within the scaffold and the cell density is low in proportion to the volume occupied by the scaffold, resulting in low efficiency in the number of cells transferred during transplantation.In addition, there are problems in that the remaining scaffold is gradually degraded, which can cause structural deformation and changes in tissue continuity.

[0025] However, despite the above problems, cell sheets composed of a single layer of cells through 2D culture without a support have the disadvantages of being weak and therefore easily torn, making them difficult to handle, and because they are composed of a single layer of cells, they have the limitation that it is difficult to deliver high concentrations of cells when used for transplantation.To solve these problems, there have been attempts to form a multi-layer structure to increase cell density, but the process of forming the multi-layer structure is complicated, and there are limitations in that it is difficult to achieve 100% interlayer integrity.

[0026] When cells are seeded at a high concentration to form a multi-layered cell structure, a high number of cells can be delivered per unit area. However, because the cells seeded at a high concentration aggregate locally to form spheroids, there is a limit in that a cell sheet with uniform thickness and structural continuity cannot be formed.

[0027] In contrast, the cell sheet of the present invention has been confirmed to have high cell transfer efficiency and be advantageous for tissue regeneration by forming a sheet with a high cell density composed of multiple layers of cells even without a support (Experimental Example 4, Figure 6).In addition, since a support is not required in the process of forming the cell sheet, there is no risk of structural deformation occurring during the decomposition of the support, resulting in high structural stability, and since the behavior of the cells is controlled through the composition of the medium rather than through coating, there is no need to add any additional processes, making it easy to apply as it is the same as the process for handling existing cells.

[0028] The cell sheet of the present invention has the advantage that, since the formation of the cell sheet is induced under non-adherent conditions, it is not greatly restricted by interactions between cells and a substrate, and since surface treatment of the culture vessel is not required, it is possible to construct culture vessels of various materials using 3D printing, etc. Furthermore, it was confirmed that the cell sheet of the present invention allows the formation of a cell structure composed of multiple layers of cells without a lamination process, and therefore the structural stability is high since it is not a multilayer structure due to lamination, the process is shortened since the lamination process can be omitted, and the cell layer is formed thick by itself, allowing for more stable handling (Experimental Example 3, FIG. 5).

[0029] Another aspect of the present invention provides a cell sheet produced by the above-mentioned production method.

[0030] Yet another aspect of the present invention provides a transplant for tissue repair, comprising the cell sheet.

[0031] Specifically, the tissue may be one or more selected from the group consisting of cartilage, bone, cornea, conjunctiva, adipose tissue, skin, muscle, fascia, tendon, aponeurosis, ligament, joint capsule, mucous sac, and epithelial tissue, and more specifically may mean cartilage, but is not limited thereto.

[0032] The cells used in the cell sheet may be cells differentiated from human pluripotent stem cells into cells of the desired tissue.

[0033] In the present invention, the term "tissue repair" may be used as a concept including both tissue regeneration and healing, where regeneration refers to reconstructing a damaged site with the same tissue composition as the damaged cells, and healing refers to a fibroproliferative response that patches damaged tissue.

[0034] In the present invention, the term "for tissue repair" refers to any application for repairing damaged tissue, including, but not limited to, cartilage damage, muscle or tendon damage, cosmetic filler, plastic prosthesis, etc.

[0035] Cartilage and soft tissues do not regenerate or heal on their own after being damaged, and therefore, unlike other tissues, methods to promote tissue growth are not suitable for use, and transplant-based treatments are known to be effective.

[0036] The "graft material for tissue repair" of the present invention means a composition that uses physical properties to fill the damaged area when tissue such as cartilage, bone, cornea, conjunctiva, adipose tissue, skin, muscle, fascia, tendon, aponeurosis, ligament, joint capsule, mucous sac, etc. is damaged, thereby treating the damage at the defect or depression and restoring the original volume, and is not limited to the embodiment as long as it is a composition used for the above-mentioned purpose, and can be appropriately modified and applied.

[0037] Although the cell sheet of the present invention does not include a separate support, it is composed of a high concentration of cells and has been confirmed to have excellent therapeutic and regenerative effects on the transplanted tissue site (Figure 6). A tissue repair transplant material containing the cell sheet can exhibit high tissue regeneration efficiency. Effect of the Invention

[0038] The cell sheet manufacturing method of the present invention is characterized in that it is possible to form a cell sheet in a non-adherent state without a support, and that it is detachable and easy to handle without a separate coating process. Furthermore, it is possible to manufacture a cell sheet that can transplant a high number of cells per unit area without a separate lamination or multi-layering process.

[0039] The effects of the present invention are not limited to the effects described above, but should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description of the present invention or the claims. [Brief description of the drawings]

[0040] [Figure 1] FIG. 1 is a diagram showing a method for producing a cell sheet using hyaluronic acid (HA) and PEG (polyethylene glycol). [Diagram 2] FIG. 2 shows the results of confirming the degree of cell aggregates in the cell sheets produced by the production methods of Example 1 and Comparative Examples 1 to 3 after 24 hours of culture. [Diagram 3] FIG. 2 is a diagram showing the results of observing the number of nodules in Example 1 and Comparative Examples 1 to 3. [Figure 4] FIG. 2 is a diagram showing the results of observing the sizes of nodules in Example 1 and Comparative Examples 1 to 3. [Diagram 5] FIG. 2 is a diagram showing the process of attaching the cell sheet of Example 1 to a PLA (Poly Lactic Acid) mesh. [Figure 6]The cell sheet was attached to a PLA mesh in a sandwich shape, and then chondrocyte differentiation was induced for 2 to 3 weeks. The morphology of the formed tissue and the presence or absence of matrix formation were confirmed through histological staining. (A: cell sheet of Example 1; B: cell sheet seeded at high concentration on the surface of a collagen gel sheet) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The present invention will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present invention, and the present invention is not limited to the following examples.

[0042] Example 1. Manufacturing method of cell slap using HA and PEG

[0043] A method for producing a cell sheet using hyaluronic acid (HA) and polyethylene glycol (PEG) is simply shown in FIG.

[0044] Specifically, the bottom surface of a square well with four closed sides was coated with poly-HEMA (poly(2-hydroxyethyl methacrylate)) to prevent cell attachment.

[0045] Rabbit meniscus-derived chondrocytes (passages 3–5) were cultured at 2 × 10 in growth medium containing 1 mg / ml hyaluronic acid (HA) while preventing cell attachment to the bottom surface of the well. 6 The cells were seeded at a concentration of 100 μl / 100 μl and cultured for 2.5 hours to induce the cell layer to sink uniformly. The growth medium was α-MEM (alpha-Minimum Essential Medium, Gibco Invitrogen) medium supplemented with 5% fetal bovine serum (FBS, Lonza), 1% penicillin / streptomycin (Welgene), 2 mM L-glutamine (Welgene), 10 -8M dexamethasone (Sigma Aldrich) and 10 -4 M ascorbic acid (Sigma Aldrich) was added to prepare the solution.

[0046] Thereafter, a growth medium containing 1% polyethylene glycol (PEG) was added and the cells were cultured to produce the cell sheet of Example 1.

[0047] Comparative Example 1. Production of cell sheets treated with growth medium only

[0048] The cell sheet of Comparative Example 1 was produced by the production method of Example 1, while maintaining all the same conditions as in Example 1, except that hyaluronic acid (HA) and polyethylene glycol (PEG) were not added.

[0049] Comparative Example 2. Production of cell sheets treated with HA only

[0050] The cell sheet of Comparative Example 2 was produced using the manufacturing method of Example 1, but the use of a growth medium containing hyaluronic acid (HA) was maintained the same as in Example 1. After culturing for 2.5 hours, a growth medium not containing polyethylene glycol (PEG) was added to produce the cell sheet of Comparative Example 2.

[0051] Comparative Example 3. Production of cell sheets treated with PEG only

[0052] The cell sheet of Comparative Example 3 was produced by maintaining all the conditions identical to those of Example 1, but using a growth medium without hyaluronic acid (HA) when initially seeding chondrocytes derived from rabbit meniscus.

[0053] The manufacturing methods of Example 1 and Comparative Examples 1 to 3 are simply shown in FIG.

[0054] Experimental Example 1. Morphological analysis of cell sheets with and without HA and PEG

[0055] In order to analyze the morphology of cell sheets containing or not containing hyaluronic acid (HA) and polyethylene glycol (PEG), it was confirmed whether the cell sheets prepared by the above-mentioned preparation method had a surface of uniform thickness without cell aggregate phenomenon.

[0056] Specifically, the cell sheets produced by the production methods of Example 1 and Comparative Examples 1 to 3 were subjected to culture conditions and the degree of cell aggregates after 24 hours were confirmed, and the results are shown in Figure 2.

[0057] As a result, as shown in Figure 2, it was confirmed that the presence or absence of hyaluronic acid (HA) at a concentration of 1 mg / ml at the time of initial cell seeding and the presence or absence of 1% PEG in the medium at the time of medium addition each significantly affected the degree of cell aggregation.

[0058] Specifically, in Comparative Example 1, which did not contain either HA or PEG, after 24 hours of culture, numerous low-density spheroids were formed, similar to general spheroid formation conditions. In Comparative Example 2, in which HA was included at the time of cell seeding, there was a strong tendency to form relatively dense spheroids. In Comparative Example 3, in which only a medium containing PEG was added without HA, cells aggregated in a disorderly manner, forming giant spheroids.

[0059] On the other hand, in Example 1, where cells were seeded in the presence of HA and medium containing PEG was added, cell aggregation was minimized and spheroids were not formed, but the cells remained at the bottom of the culture medium, maintaining a sheet shape and a relatively uniform thickness.

[0060] These results may be due to the differential effects of the two hydrogel components present in the culture medium, which modulated cell behavior and thereby altered the tendency of cells to aggregate and form spheroids.

[0061] In other words, HA, which has hydrogel properties, distributes the medium components evenly and maintains intercellular bonds, while PEG contained in the medium exerts a repulsive force on the cells, which generates a force that tries to minimize the interface between the cells and PEG contained in the medium, thereby maintaining the shape of the cell slap.

[0062] Experimental Example 2. Confirmation of the degree of nodule formation depending on whether or not HA and PEG are included

[0063] In order to confirm the degree of nodule formation depending on whether or not HA and PEG are included, the number and size of the nodules in Example 1 and Comparative Examples 1 to 3 were observed using the same method as in Experimental Example 1. The results of confirming the number are shown in FIG. 3, and the results of confirming the size are shown in FIG. 4.

[0064] As a result, as shown in Figure 3, the number of aggregates formed was the highest in Comparative Example 1, where no treatment was performed, and the numbers of aggregates formed were lower in the hyaluronic acid only treatment group (Comparative Example 2), in which relatively large spheroids were formed, and the PEG only treatment group (Comparative Example 3), in which cell aggregation occurred in a disordered manner, compared to Comparative Example 1. It was confirmed that the number of aggregates formed was the lowest in Example 1, in which both HA and PEG were treated.

[0065] In addition, as shown in FIG. 4, it was confirmed that the size of the aggregates in Comparative Example 1, which was untreated, and Example 1, in which both HA and PEG were treated, was significantly smaller than those in Comparative Examples 2 and 3.

[0066] Considering the above results, it was confirmed that the number of aggregates formed was the smallest and the size was the smallest in Example 1, where both HA and PEG were treated. This result is very important for overcoming the technical limitations that existed in the past.

[0067] Existing cell sheets based on a single cell layer have limitations in that they have weak physical strength and a low cell concentration when used for transplantation. In order to overcome the limitations of cell sheets based on a single cell layer, a cell sheet composed of multiple cells and a method for manufacturing the same have been sought. However, attempts to form a cell sheet composed of multiple cells by seeding cells at a high concentration have failed to solve the problem that the higher the cell concentration, the more aggregation occurs and the more spheroids are formed. Therefore, in order to manufacture a cell sheet composed of multiple cells, a separate lamination process is required, which is structurally unstable and requires a long process.

[0068] However, by using the method for producing a cell sheet containing HA and PEG of the present invention, the problem of excessive spheroid formation can be resolved, making it possible to produce a cell sheet composed of multiple layers of cells without a separate layering process.

[0069] Experimental Example 3. Confirmation of the usability of cell sheets produced containing HA and PEG

[0070] In using the cell sheet prepared in Example 1, it was confirmed whether the limitations and inconveniences present in the process of using cell sheets prepared by the existing cell sheet manufacturing methods were overcome.

[0071] When producing a cell sheet using an existing scaffold, it is difficult to separate the cell sheet cleanly without a separate processing step, and furthermore, there is a possibility that the structure of the cell sheet may be deformed and the tissue continuity may change during the process of decomposing the scaffold or separating it from the scaffold.

[0072] In addition, existing cell sheets based on a single cell layer have the disadvantage that they are weak and therefore easily torn, making them difficult to handle without a separate handling tool. When forming a multi-layer structure, not only is the process very complicated, but it is also difficult to achieve 100% interlayer integrity, making it impossible to form a cell sheet with uniform thickness and structural continuity.

[0073] The cell sheet of the present invention is characterized in that it is formed under non-adherent conditions without a support and is formed to a sufficient thickness by being composed of multiple layers of cells without a separate layering process. To confirm this, a process of attaching the cell sheet of Example 1 to a PLA (Poly Lactic Acid) mesh was demonstrated (Figure 5).

[0074] As shown in FIG. 5, the cell sheet was formed wider than the PLA mesh, and then wrapped around and attached to the front and back of the collagen-coated PLA mesh to form a complex for chondrocyte differentiation.

[0075] At this time, since the cell sheet of Example 1 was formed under non-adhesive conditions without a separate support, it was possible to separate a uniform cell sheet with a certain thickness using only a cell scraper.

[0076] In addition, the separated cell sheet was formed thick enough with multiple layers of cells, so it was confirmed that it could be handled without tearing using only tweezers without any additional handling tools.

[0077] In other words, the cell sheet produced by the cell sheet production method of the present invention is formed under non-adhesive conditions without a support, and it was confirmed that the cell sheet is not only structurally stable, but also has uniform thickness and structural continuity by forming a multi-layered cell structure independently without a separate layering process.

[0078] Experimental Example 4. Confirmation of the effect of matrix formation of cell sheets produced containing HA and PEG

[0079] In order to confirm the matrix formation effect of the cell sheet produced by the production method of the present invention, the cell sheet was attached to a PLA mesh in a sandwich shape, and it was confirmed whether the cells on both surfaces of the mesh bonded with each other and integrated into a single tissue, and the efficacy of matrix formation during cartilage differentiation.

[0080] Specifically, the cell sheet seeded at a high concentration on the surface of the collagen gel sheet and the cell sheet of Example 1 were attached to a PLA mesh in a sandwich shape, and chondrocyte differentiation was induced for 2 to 3 weeks. Thereafter, the morphology of the formed tissue and the presence or absence of matrix formation were confirmed by histological staining (FIG. 6).

[0081] Specifically, the fixed tissues were paraffin sectioned to a thickness of 5-7 μm, dried, stained with hematoxylin and eosin (H&E), and observed under a high-resolution optical microscope to confirm the morphology of the tissues. To confirm the formation of a cartilage-specific matrix through Safranin O staining, nuclei were stained with Weigert's hematoxylin for 5 minutes and then washed with distilled water for 10 minutes. The tissues were adapted to the ethanol solution by immersing in a 70% ethanol solution, stained with 0.02% fast green for 5 minutes, and washed with 1% acetic acid. After staining with 0.1% aqueous Safranin O for 5 minutes, the tissues were dehydrated by adapting to 70%, 80%, 90%, and 100% alcohol in sequence, adapted in xylene, and mounted with plastic mounting solution.

[0082] To confirm whether or not the collagen gel sheet remained by staining the fibrous matrix proteins using the trichrome staining method, the nuclei were stained with Weigert's hematoxylin for 10 minutes, then washed with distilled water for 10 minutes, and the cytoplasm was stained with Biebrich scarlet acid red fuchsin aqueous solution for 5 minutes. The fibrous matrix was stained with aniline blue, and the stain was fixed with 1% acetic acid, dehydrated by adapting to 70%, 80%, 90%, and 100% alcohol in sequence, and then adapted to xylene, and mounted with plastic mounting solution (Permount, Fisher Chemical). All reagents were from Sigma-Aldrich unless otherwise indicated.

[0083] As a result, as shown in Figure 6A, the tissue formed from the cell sheet of Example 1 penetrated the PLA mesh located in the center and then bonded to each other to form a single tissue, confirming that the formation of a cartilage-specific matrix occurred successfully.

[0084] On the other hand, as shown in Figure 6B, in the cell sheet formed using the collagen gel sheet, the cells seeded on the collagen surface penetrated or migrated into the PLA mesh to a significantly lower extent, forming two separate layers with the PLA mesh as the boundary. In addition, compared to the cell sheet of Example 1, the degree of cartilage matrix formation was very low, and it was confirmed that the collagen gel sheet seeded with the cells continued to remain (results of trichrome staining).

[0085] From the above results, it was confirmed that the cell sheet of the present invention prepared containing HA and PEG is formed without a separate support such as a collagen gel sheet and has a high cell density, resulting in high cell transfer efficiency and being advantageous for tissue regeneration, as well as having perfect integrity and high structural continuity.

[0086] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. For example, each component described as a single type may be implemented in a distributed form, and similarly, each component described as a distributed type may be implemented in a combined form.

[0087] The scope of the present invention is defined by the claims set forth below, and all modifications and variations that fall within the meaning, scope, and equivalent concepts of the claims should be construed as being included within the scope of the present invention.

Claims

1. a) sedimenting and culturing the cells in a growth medium containing hyaluronic acid; b) adding a growth medium containing polyethylene glycol, The method for producing a cell sheet, wherein the cells are one or more selected from the group consisting of epidermal cells, fibroblasts, hepatocytes, mesodermal stem cells, and chondrocytes.

2. The method for producing a cell sheet according to claim 1 , wherein the cell sheet is in the form of a plate.

3. The method for producing a cell sheet according to claim 1 , wherein the cell sheet is one in which cell aggregation is suppressed.

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