CELL SHEET COMPRISING HYALURONIC ACID AND POLYETHYLENE GLYCOL, AND METHOD FOR PRODUCING SAME

Abstract

Disclosed is a method of constructing a cell sheet using only cells without a support. More particularly, a method of manufacturing a multi-layered cell sheet without a separate lamination step and a cell sheet manufactured by the method are disclosed.

Claims

1. A method of manufacturing a cell sheet, the method comprising: a) a step of sedimenting and culturing cells in a growth medium comprising hyaluronic acid; and b) a step of adding a growth medium comprising polyethylene glycol.

2. The method according to claim 1, wherein the cells are one or more selected from the group consisting of epidermal cells, fibroblasts, hepatocytes, mesodermal stem cells, and chondrocytes.

3. The method according to claim 1, wherein the cell sheet has a plate shape.

4. The method according to claim 1, wherein the cell sheet has suppressed cell aggregation.

5. A cell sheet manufactured according to claim 1.

6. A graft material for tissue repair, comprising the cell sheet according to claim 5.

7. The graft material according to claim 6, wherein the tissue type is one or more selected from the group consisting of cartilage, bone, cornea, conjunctiva, adipose tissue, skin, muscle, fascia, tendon, aponeurosis, ligament, joint capsule, bursa, and epithelial tissue.

Description

DESCRIPTION OF DRAWINGS

[0040] FIG. 1 schematically illustrates a method of manufacturing a cell sheet using hyaluronic acid (HA) and polyethylene glycol (PEG).

[0041] FIG. 2 illustrates cell aggregation degrees of cell sheets manufactured by methods of Example 1 and Comparative Examples 1 to 3 after culturing for 24 hours.

[0042] FIG. 3 illustrates the number of nodules observed in each of Example 1 and Comparative Examples 1 to 3.

[0043] FIG. 4 illustrates the size of nodules observed in each of Example 1 and Comparative Examples 1 to 3.

[0044] FIG. 5 illustrates a process of attaching a cell sheet of Example 1 to a Poly Lactic Acid (PLA) mesh.

[0045] FIG. 6 illustrates the shape of tissue formed after attaching a cell sheet to a PLA mesh in the form of a sandwich, and then inducing differentiation into cartilage for 2 or 3 weeks; and a matrix observed through histological staining.

BEST MODE

[0046] Hereinafter, the present invention is described in detail with reference to examples. However, the following examples are only for aid in understanding of the present disclosure, and the present invention is not limited to the following examples.

Example 1

Method of Manufacturing Cell Sheet Including HA and PEG

[0047] A method of manufacturing a cell sheet using hyaluronic acid (HA) and polyethylene glycol (PEG) is briefly illustrated in FIG. 1.

[0048] Specifically, a bottom surface of a rectangular well whose all sides were closed was coated with poly-HEMA (poly(2-hydroxyethyl methacrylate) to prevent cell binding.

[0049] In a state where binding of cells to the bottom surface of the well is prevented, chondrocytes derived from rabbit meniscus between 3 and 5 passages were seeded in a growth medium containing 1 mg/ml hyaluronic acid (HA) at a concentration of 2×10.sup.6/100 μl and cultured for 2.5 hours to induce a cell layer to uniformly sink. Here, the growth medium was prepared by adding 5% fetal bovine serum (FBS, Lonza), 1% penicillin/streptomycin (Welgene), 2 mM L-glutamine (Welgene), 10.sup.−8M dexamethasone (Sigma Aldrich) and 10.sup.−4M ascorbic acid (Sigma Aldrich) to alpha-Minimum Essential Medium (α-MEM, Gibco Invitrogen).

[0050] Next, 1% polyethylene glycol (PEG) was added to the growth medium and cells were additionally cultured therein. As a result, a cell sheet of Example 1 was produced.

Comparative Example 1

Production of Cell Sheet Treated With Only Growth Medium

[0051] A cell sheet of Comparative Example 1 was manufactured under the same conditions as in Example 1 except that hyaluronic acid (HA) and polyethylene glycol (PEG) were not added.

Comparative Example 2

Production of Cell Sheet Treated With HA Only

[0052] A cell sheet of Comparative Example 2 was manufactured in the same manner as in Example 1 using hyaluronic acid (HA) containing a growth medium, except that a growth medium excluding polyethylene glycol (PEG) was added after culturing for 2.5 hours.

Comparative Example 3

Production of Cell Sheet Treated With PEG Only

[0053] A cell sheet of Comparative Example 3 was manufactured under the same conditions as in Example 1, except that a growth medium excluding hyaluronic acid (HA) was used when initially seeding chondrocytes derived from rabbit meniscus.

[0054] The manufacturing methods of Example 1 and Comparative Examples 1 to 3 are briefly illustrated in FIG. 1.

Experimental Example 1

Analysis of Cell Sheet Shapes Dependent Upon Presence or Absence of HA and PEG

[0055] To analyze whether the shape of the cell sheet depends upon the presence or absence of hyaluronic acid (HA) and polyethylene glycol (PEG), it was confirmed whether the cell sheets manufactured by the above methods had a uniform surface thickness without cell aggregation.

[0056] Specifically, the degree of cell aggregation was checked 24 hours after maintaining the culture state for the cell sheets manufactured by the methods of Example 1 and Comparative Examples 1 to 3. Results are illustrated in FIG. 2.

[0057] As a result, it was confirmed that whether hyaluronic acid (HA) at a concentration of 1 mg/ml was included in the initial cell seeding and whether 1% PEG was included in a medium when the medium was added greatly affected the degree of aggregation of cells, as shown in FIG. 2.

[0058] Specifically, in the case of Comparative Example 1 in which both HA and PEG were excluded, a large number of spheroids of low density were formed after 24 hours of culture similarly to general spheroid formation conditions. In the case of Comparative Example 2 containing HA at the time of cell seeding, the tendency to form relatively dense spheroids was high. In the case of Comparative Example 3 using a medium containing only PEG without HA, aggregation of cells occurred in a disorderly manner to form huge spheroids.

[0059] On the other hand, it was confirmed that in the case of Example 1 wherein cells were seeded under a condition containing HA and a medium containing PEG was added, cell aggregation was minimally suppressed so that spheroids did not appear, and the cells maintained a sheet shape in a state of being sedimented on the bottom in the culture medium and maintained a relatively uniform thickness.

[0060] This result seems to be because the spheroid formation tendency induced by cell aggregation was changed by controlling the behavior of cells by the differential action of the two hydrogel components present in the culture medium.

[0061] That is, it seems that, while HA having the characteristics of a hydrogel maintains intercellular bonding while evenly distributing the medium components, PEG contained in the medium exhibits repulsive force against cells, so that a force to minimize the interface between PEG and cells contained in the medium is generated, thereby maintaining the shape of the cell sheet.

Experimental Example 2

Confirmation of Nodule Formation Dependent Upon Presence or Absence of HA and PEG

[0062] To investigate the degree of nodule formation dependent upon the presence or absence of HA and PEG in the same manner as in Experimental Example 1, the number and sizes of nodules of each of Example 1 and Comparative Examples 1 to 3 were observed. Results of the number of the nodules are illustrated in FIG. 3, and results of the size thereof are illustrated in FIG. 4.

[0063] As shown in FIG. 3, it was confirmed that the number of nodules was the highest in the untreated Comparative Example 1, the number of nodules formed in the group treated with only hyaluronic acid (Comparative Example 2) in which relatively large spheroids were formed; and the group treated with only PEG (Comparative Example 3) in which cell aggregation occurred in a disorderly manner was smaller than that of Comparative Example 1, and the number of nodules formed in Example 1 treated with both HA and PEG was the smallest.

[0064] In addition, it was confirmed that the sizes of nodules in the untreated Comparative Example 1, and Example 1 treated with both HA and PEG were significantly smaller than those of Comparative Examples 2 and 3, as shown in FIG. 4.

[0065] When the above results are taken together, the number of aggregates formed in Example 1 treated with both HA and PEG was the smallest and the size thereof was also the smallest. This result corresponds to a very important content for overcoming the technical limitations existing in the prior art.

[0066] Existing single cell layer-based cell sheets have weak physical strength and limitations in that the cell concentration is low when used for transplantation. To overcome the limitations of such a single cell layer-based cell sheet, a cell sheet composed of multi-layered cells and a method of manufacturing the same have been required. However, attempts to form a cell sheet composed of multi-layered cells by seeding cells at a high concentration do not solve the problem because a large number of spheroids are formed due to cell aggregation increasing as the concentration of cells increased. Accordingly, a separate lamination process is required to manufacture a cell sheet composed of multi-layered cells, which causes structural instability and process extension.

[0067] However, the problem of excessive spheroid formation can be addressed by using the method for manufacturing a cell sheet containing HA and PEG of the present invention, so that a cell sheet composed of multi-layered cells can be manufactured without a separate lamination process.

[0068] Experimental Example 3

Confirmation of Usability of Cell Sheet Including HA and PEG

[0069] It was confirmed whether the limitations and inconveniences existing in a process of using a cell sheet manufactured by an existing cell sheet manufacturing method can be overcome by using the cell sheet manufactured in Example 1.

[0070] In the case of manufacturing a cell sheet using an existing support, it is difficult to cleanly separate the cell sheet without a separate treatment process, and furthermore, there is a possibility of structural deformation of the cell sheet and a possibility of change in tissue continuity during the disassembly of the support or separation from the support.

[0071] In addition, in the case of existing single cell layer-based cell sheets, there is a disadvantage that they are easily torn due to weak strength and it is difficult to handle the sheets without a separate handling tool. In the case of forming a multilayer structure, the process is very complicated, it is difficult to implement 100% interlayer integrity, and it is impossible to form a cell sheet having a uniform thickness and structural continuity.

[0072] The cell sheet of the present invention is characterized by being formed under a non-adherent condition without a support, being composed of multi-layered cells without a separate lamination process, and being formed to be sufficiently thick. To confirm such characteristics, a process of attaching the cell sheet of Example 1 to a Poly Lactic Acid (PLA) mesh was demonstrated (FIG. 5).

[0073] As shown in FIG. 5, the cell sheet was formed to be wider than the PLA mesh, and then attached around the front and back of a collagen-coated PLA mesh, thereby forming a complex for cartilage differentiation.

[0074] Here, since the cell sheet of Example 1 was formed under a non-adherent condition without a separate support, it was possible to separate a uniform cell sheet having a constant thickness only with a cell scraper.

[0075] In addition, it was confirmed that since the separated cell sheet was formed to be sufficiently thick with multi-layered cells, it was possible to handle without tearing only with tweezers without a separate handling tool.

[0076] That is, it was confirmed that since the cell sheet manufactured by the method of manufacturing a cell sheet of the present invention is formed under a non-adherent condition without a support, the multi-layered cell structure is formed by itself without a separate lamination process, so that the multi-layered cell structure is structurally stable and has uniform thickness and structural continuity.

Experimental Example 4

Confirmation of Matrix Formation Effect of Cell Sheet Prepared in the Presence of HA and PEG

[0077] To investigate the matrix-forming effect of the cell sheet manufactured by the method of the present invention, the cell sheet was attached to a PLA mesh in a sandwich form. Whether cells on both surfaces of the mesh were combined with each other and integrated into a single tissue was investigated, and the efficacy of matrix formation during cartilage differentiation was investigated.

[0078] Specifically, a cell sheet seeded at a high concentration on a surface of a collagen gel sheet and the cell sheet of Example 1 were respectively attached to the PLA mesh in a sandwich form, and then cartilage differentiation was induced for 2 or 3 weeks. Next, The shapes of the formed tissues and matrix formation were confirmed through histological staining (FIG. 6).

[0079] Specifically, the fixed tissue was paraffin-sectioned to a thickness of 5 to 7 μm and dried, and then subjected to hematoxylin/eosin staining (H&E staining), and then the tissue shape was observed with a high-resolution optical microscope. To confirm the formation of a cartilage-specific matrix through Safranin O staining, the nucleus was stained with Weigert's hematoxylin for 5 minutes, and then washed with distilled water for 10 minutes. The tissue was immersed in 70% ethanol solution to adapt to the ethanol solution, and then 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 sequentially adapting to 70%, 80%, 90%, and 100% alcohol, and then placed in xylene to adapt, and then mounted with a plastic mounting solution (mountant).

[0080] To investigate whether the collagen gel sheet remains by staining the fibrous matrix protein by the Trichrome staining method, the nucleus was stained with Weigert's hematoxylin for 10 minutes, and then washed with distilled water for 10 minutes, and the cytoplasm was stained with an aqueous solution of Biebrich scarlet acid red fuchsin for 5 minutes. After staining the fibrous substrate with aniline blue, the tissue was stained and fixed with 1% acetic acid, dehydrated by sequentially adapting to 70%, 80%, 90%, and 100% alcohol, and placed in xylene to adapt, and then mounted with a plastic mounting solution (mountant). All reagents were purchased from Sigma Aldrich unless otherwise indicated.

[0081] As a result, as illustrated in FIG. 6A, it was confirmed that the tissues formed from the cell sheet of Example 1 were combined with each other after penetration into the PLA mesh located in the center to form a single tissue, and the formation of the cartilage-specific matrix was excellent.

[0082] On the other hand, in the cell sheet formed using the collagen gel sheet, the degree of penetration or migration of cells seeded on the collagen surface into the PLA mesh was remarkably low, so that two separate layers were formed with the PLA mesh as a boundary, as illustrated in FIG. 6B. In addition, compared with the cell sheet of Example 1, it was confirmed that the degree of cartilaginous matrix formation was very low, and the cell-seeded collagen gel sheet continued to remain (trichrome staining result).

[0083] From the above results, it was confirmed that the cell sheet of the present invention including HA and PEG was formed without a separate support such as a collagen gel sheet and exhibited high structural continuity with perfect integrity as well as high cell transfer efficiency and advantageous tissue regeneration due to high cell density.

[0084] The aforementioned description of the present invention is provided by way of example and those skilled in the art will understand that the present invention can be easily changed or modified into other specified forms without change or modification of the technical spirit or essential characteristics of the present invention. Therefore, it should be understood that the aforementioned examples are only provided by way of example and not provided to limit the present invention. For example, each of constituents described as a single form may be separately implemented and, similarly, constituents described as being separated may be implemented in a combined form.

[0085] It should be understood that the scope of the present invention is defined by the following claims and the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims.