WO2000030695A1

WO2000030695A1 - Mucosal epithelial cell sheet and method for producing the same

Info

Publication number: WO2000030695A1
Application number: PCT/JP1999/006517
Authority: WO
Inventors: Minoru Ueda
Original assignee: Japan Tissue Engineering Co., Ltd.
Priority date: 1998-11-26
Filing date: 1999-11-22
Publication date: 2000-06-02
Also published as: JP3043727B1; AU1186000A; JP2000157624A

Abstract

A mucosal epithelial cell sheet consisting of a plural number of layers of oral inner marginal mucosal epithelial cells which adhere to teeth continuously to oral mucosal epithelial cells so as to form a part of the gingival trough. Because of having a potent ability to divide and a low antigenicity, these oral inner marginal mucosal epithelial cells can be prepared within a short time and take over a long period of time.

Description

Description Mucosal epithelial cell sheet and method for producing the same

The present invention relates to a mucosal epithelial cell sheet and a method for producing the same, and more particularly to a mucosal epithelial cell sheet formed by forming a plurality of layers of mucosal epithelial cells and a method for producing the same. Landscape technology

Living cells and tissues may be transplanted or introduced into living organisms, including humans.

For example, tissue may be implanted as a wound dressing. In this case, the part where the tissue of the living body has been partially removed by surgery or accident is replaced with another tissue (graft) to supplement the removed part or improve the appearance. Various implants have been used for such purposes.

In particular, a multi-layered sheet of epidermal cells has the advantage that it can be prepared from its own epidermal cells. There is no rejection reaction in the epidermal cell sheet thus produced, and therefore, the epidermal cell sheet is highly evaluated as a useful explant for burns and the like.

In addition, epithelial cells isolated from oral mucosal cells have a shorter metabolic cycle than epidermal cells because keratinocytes of the skin, that is, epidermal cells are also unseparated cells. It also has the advantage that it can be maintained for a long period of time without squaring. From these properties, oral mucosal cells are considered to be more useful as tissue for transplantation than epidermal keratinocytes.

However, when transplanting a tissue into a living body, it is necessary to prepare a graft in a short time after the necessity of transplantation. Especially in the case of severe burns, it may take time to prepare the implant. On the other hand, non-self transplants, which were substituted in cases where transplants could not be made from their own tissues, were rejected after transplantation and once survived Even they are eliminated in a short time.

Accordingly, it is an object of the present invention to provide an implant that can be prepared more quickly and that can survive for a longer period.

It is also an object of the present invention to more rapidly prepare a graft that can survive over a long period of time. Disclosure of the invention

The mucosal epithelial cell sheet of the present invention is constituted by forming a plurality of layers of the inner lining mucosal epithelial cells that adhere to the teeth continuously to the oral mucosal epithelial cells and form a part of the gingival sulcus. It is characterized in that it is a treated mucosal epithelial cell sheet.

In another embodiment, the above-mentioned mucosal epithelial cell sheet of the present invention is characterized in that the oral mucosal epithelial cell is derived from one that has adhered to a tooth during tooth extraction and has been separated from gingiva.

Further, the method for producing a mucosal epithelial cell sheet of the present invention is characterized in that the oral mucosal epithelial cells forming a part of the gingival sulcus, which adhere to the teeth continuously with the oral mucosal epithelial cells, form a plurality of layers. A method for producing a mucosal epithelial cell sheet constituted by the method described above, comprising: obtaining the oral intraoral mucosal epithelial cells; and supporting cells that support the growth of the oral intraoral mucosal epithelial cells; Culturing the oral mucosal epithelial cells in the presence of the supporting cells to obtain a formed mucosal epithelial cell sheet in order to form a mucosal epithelial cell sheet composed of mucosal epithelial cells. The method is characterized by a method for producing a mucosal epithelial cell sheet.

In one aspect of the present invention, the method for producing a mucosal epithelial cell sheet is characterized in that the oral rim mucosal epithelial cells are separated from the gingiva while being attached to the teeth during tooth extraction.

In another aspect of the present invention, the method for preparing a mucosal epithelial cell sheet comprises dividing the supporting cells by irradiation with a drug or radiation before co-culturing the supporting cells and the oral mucosal epithelial cells. It is characterized in that it includes the loss of performance. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a sectional view of a tooth and a gingiva.

FIG. 2 is a partially enlarged view of FIG. 1 showing the position of the oral mucosal epithelial cell layer.

FIG. 3 is a graph showing the ability of oral mucosal epithelial cells to form a knee.

FIG. 4 is a graph showing the growth curve of the oral mucosal epithelial cells.

FIG. 5 is a graph showing the growth of oral mucosal epithelial cells.

FIG. 6A is a graph showing the antigenicity of oral mucosal epithelial cells, and FIG. 6B is a graph showing the antigenicity of oral rim mucosal epithelial cells.

Fig. 7A is an electron micrograph of a mucosal epithelial cell sheet composed of oral mucosal epithelial cells, Fig. 7B is an electron micrograph of a cell sheet composed of oral mucosal epithelial cells, Fig. 7C 1 is an electron micrograph of a cell sheet composed of epidermal cells. FIG. 8 is a graph showing the engraftment rate of the mucosal epithelial cell sheet formed from the oral mucosal epithelial cells. Detailed description of the invention

The mucosal epithelial cell sheet of the present invention is composed of a plurality of layers of oral mucosal epithelial cells.

Oral mucosal epithelial cells are a type of oral mucosal epithelial cells. As shown in FIGS. 1 and 2, the oral mucosal epithelial cells constitute an inner epithelial cell layer 14 which is a part of the gingiva 12 supporting the lower end of the tooth 10. In addition, the oral mucosal epithelial cells adhere to the surface of the enamel layer 16 of the tooth 10 and form a gingival sulcus 18 generally called a periodontal pocket. In the present specification, the oral rim mucosal epithelial cells mean, for convenience, a cell group specified at an anatomical position in a living body.

As shown in FIG. 2, at the upper end of the gingiva 12, the lining epithelial cell layer 14 is continuous with the oral mucosal cell layer 20, and within the gingiva 12, there is a connective tissue portion 22. It is located. The oral mucosal cell layer 20 is composed of oral mucosal epithelial cells different from the oral mucosal epithelial cells.

The present inventor has found that the oral mucosal epithelial cells have different properties from the oral mucosal epithelial cells arranged on the outer side surface of the gingiva 12 and other parts of the oral cavity. In other words, in general, plaque tends to accumulate in the gingival sulcus 18 and bacteria easily proliferate, so the oral lining mucosal epithelial cells in the lining epithelial cell layer 14 are always at risk of bacterial infection . For this reason, it is considered that the oral mucosal epithelial cells are specialized to cope with such an environment, unlike similar mucosal epithelial cells.

In particular, it was found that the lining epithelial cells of the oral cavity had much higher dividing ability than the oral mucosal epithelial cells of other sites. This seems to be against bacterial infection and damage. It was also found that the antigenicity was lower than that of oral mucosal epithelial cells at other sites. That is, oral mucosal epithelial cells have the advantage that rejection is less likely to occur than oral mucosal epithelial cells.

Therefore, the mucosal epithelial cell sheet of the present invention has the above-mentioned characteristics of the oral marginal mucosal epithelial cell sheet as it is, and has higher division ability and lower antigenicity than the conventional oral mucosal epithelial cell sheet. In addition, it can be prepared in a short period of time and achieve long-term engraftment.

In the living body, the inner epithelial cell layer 14 composed of the oral inner marginal epithelial cells is continuous with the oral mucosal cell layer 20 of the gingiva 12 as shown in FIG. The inner mucosal epithelial cells are attached to the enamel layer 16 of the tooth 10 on the side of the tooth 10, and the inner mucosal epithelial cells are converted to the oral mucosal cells at the upper end of the gingival sulcus 18. It can be easily isolated by separating it from the layer 20 and detaching it from the enamel layer 16.

The method of separating the oral mucosal epithelial cells from the oral mucosal cell layer 20 can be performed by ordinary excision, etc., but the degree of adhesion to the enamel layer 16 depends on the degree of adhesion to the oral mucosal cell layer 20 and connective tissue. Since it is stronger than continuity, it can be separated from the oral mucosal cell layer 20 and the connective tissue part 22 while being attached to the enamel layer 16 when the tooth 10 is extracted. Therefore, it can be easily collected from the extracted tooth 10.

In most cases, tooth extraction is performed due to abnormalities / problems in the tooth 10, so the oral rim mucosal epithelial cells themselves attached to and recovered from the tooth 10 retain the characteristics of the cells as they are . Therefore, it is advantageous to use such oral mucosal epithelial cells.

The collected inner lining mucosal epithelial cells contain, for example, connective tissue ゃ debris. In such cases, the enzyme can be removed using a suitable enzyme such as dispase, trypsin, or nylon mesh.

The mucosal epithelial cell sheet of the present invention has properties of oral mucosal epithelial cells, and also retains the structural characteristics of oral mucosal epithelial cells. For example, it is known that oral mucosal epithelial cells have a wider cell gap than oral mucosal epithelial cells at other sites. For this reason, the mucosal epithelial cell sheet of the present invention can be easily specified based on the enlarged cell gap. The form other than these features is the same as other mucosal cell sheets, and can be transplanted to a site where the mucosal cell sheet can be transplanted without a sense of incongruity.

The mucosal epithelial cell sheet of the present invention is produced by co-culture with a supporting cell. Supporting cells include, for example, fibroblasts, for example, mouse NIH 3T3 cells, 3T3J2, and Swiss 3T3.From the viewpoint of the thickness and growth rate of the formed mucosal epithelial cell sheet, 3 T3J2 cells are preferred.

The supporting cells are preferably subjected to a treatment for eliminating the proliferation ability so as not to interfere with the growth of the oral mucosal epithelial cells. The disappearance of the proliferation ability can be performed by a method well known in the art, for example, treatment with mitomycin C or irradiation. In addition, culture is performed using a normal cell culture vessel such as a plastic dish.

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Propagation of the oral mucosal epithelial cells is performed in the presence of supporting cells. Since the supporting cells such as 3Τ3 form layers before the oral mucosal epithelial cells, the oral mucosal epithelial cells grow on the supporting cells even if they are seeded simultaneously with the oral mucosal epithelial cells. Therefore, the oral mucosal epithelial cells may be seeded simultaneously with the supporting cells as long as the supporting cells are present at the time of proliferation, or may be seeded thereon after the supporting cells form a layer.

Oral rim mucosal epithelial cells can be seeded at a cell density of 1 × 10 ⁴ cells / cm ² or higher, while supporting cells are 1 × 10 cells / cm ^{2 to} l × 10 ³ cells / cm 2 It can be sowed with ^two or more. If the number of supporting cells is smaller than this, it is not preferable because it cannot sufficiently serve as a supporting cell. In addition, the supporting cells are used to support the growth of the oral mucosal epithelial epithelial cells. This is not preferred because it hinders the growth of the oral mucosal epithelial cells. If the number of oral mucosal epithelial cells is less than this range, it will not be possible to efficiently proliferate, which is not preferable.

In the present invention, a growth medium is used for growing oral mucosal epithelial cells on supporting cells. The growth medium used for this purpose can be selected from various media known in the art. By culturing in a growth medium, oral lining mucosal epithelial cells readily form multiple layers. On the other hand, the supporting cells die only by supporting the growth of the mucosal epithelial cells, and are no longer present when the mucosal epithelial cells form multiple layers.

It is preferable to use an epithelial sheet forming medium (EFM) capable of forming a sheet as the growth medium. EFM is a medium that is usually used to grow epidermal cells in layers. It consists of a 3: 1 mixture of DMEM and Ham's F-12 medium, and contains 5% FBS and 5 μg / ml insulin. Transferrin, 5 zg / ml, 2 xlO— ⁹ M triiodothyronine, 1 xl (T ⁹ M cholera toxin, 0.4 μg / ml Hyde mouth cortisone, 10 OU / ml penicillin, 0.1 zg / ml It is supplemented with kanamycin, 0.25 / g / ml amphotericin B and 1 Ong / ml human recombinant epidermal growth factor (EGF).

The multilayered oral mucosal epithelial cells are separated from the culture vessel while maintaining the layer structure. This separation can be performed by methods known in the art, and can be easily performed, for example, by using a suitable enzyme such as dispase to inhibit the adhesion of the basal layer.

The obtained mucosal epithelial cell sheet is transplanted into a living body by a method known in the art. Since the mucosal epithelial cell sheet of the present invention is composed of a plurality of layers of oral mucosal epithelial cells, it can be transplanted as it is to a transplant site. Further, the mucosal epithelial cell sheet of the present invention is suitable for transplantation in a humid environment such as the oral cavity and under the skin, but can also be transplanted on the epidermis in contact with the outside world. Transplantation can be performed by directly applying a known method adapted to the oral mucosal epithelial cell sheet.

In addition, the oral mucosal epithelial cells constituting the oral mucosal epithelial cell sheet include: Gene transfer can be performed in the same manner as in the case of the mucosal cells.

Gene transfer can be easily performed by introducing a gene to be transferred (target gene) by a conventional method. Since oral mucosal epithelial cells are more undifferentiated than mature cells such as epidermal cells, genes can be introduced more easily than mature cells.

Genes of interest include factors useful for increasing graft engraftment efficiency, such as antibiotics and antimicrobial peptides (such as dufensin) to prevent bacterial infection during graft engraftment, Includes factors that promote graft survival, such as platelet growth factor (PDGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HFG). These genes are known in the literature and can be easily obtained from public institutions and the like.

In addition, a gene for gene therapy can be a target gene. Target genes that can be targeted for such gene therapy include congenital and acquired deficiency factors, such as blood coagulation factors, insulin, various growth factors, tumor suppressor genes, and tumor antigen genes. These genes are easily available as described above. Example

Hereinafter, examples representing the present invention will be described.

I. Recovery of oral mucosal epithelial cells

Oral rim mucosal epithelial cells were recovered from the tooth after the tooth extraction process with the consent of a patient who came to the hospital for the purpose of wisdom tooth extraction. The oral mucosal epithelial layer was torn off at the upper end of the gingiva during tooth extraction, and thus was separated from the oral mucosal epithelial cells at other sites. The tooth extraction was performed as aseptically as possible, and the oral mucosal epithelial layer adhering to the extracted tooth was carefully separated from the tooth enamel cement transition. The oral mucosal epithelial layer was washed with a phosphate buffer containing 100 OU / ml penicillin (Sigma, St. Louis, Mo.), lmg / ml kanamycin and 2. amphotericin B (Gibco, Gland Island, NY). The solution was immersed twice in PBS (PBS) at 37 for 30 minutes. The tissue is then placed in Dulbecco's Modified Minimum Essential Medium (DMEM) containing 100 OPU / ml Dispase® (manufactured by Godo Shusei Co., Ltd.) for 16 hours. The cells were immersed at 4 ° C and then treated with 0.25% trypsin (Gibco, Gland Isl and NY) for 30 minutes at room temperature to separate the cells. Enzyme activity was stopped by washing with DMEM containing 10% fetal calf serum (FBS) (Hyclone, UT). Thereafter, the mixture was stirred in DMEM containing 10% FBS for 30 minutes, and then the debris was filtered with a 50 μm nylon gauze to isolate the oral mucosal epithelial cells.

II. Characteristics of oral mucosal epithelial cells

(1) Sheet forming ability

In order to confirm the proliferation activity of the oral mucosal epithelial cells, the above-mentioned oral mucosal epithelial cells (hereinafter referred to as inner epithelial cells) and the mucosal epithelial cells at other parts of the oral cavity (hereinafter referred to as oral epithelial cells) ) And the ability to form colonies with epidermal cells and the growth rate were measured. Oral epithelial cells were isolated from the human alveolar gingival mucosa in the oral cavity. Epidermal cells were also isolated from human abdominal skin.

The colony forming ability was determined by a known method. Briefly, it is as follows. Into a 25 cm ² T-type flask (Nunc), inoculate 1 × 10 ³ limbal epithelial cells, oral epithelial cells and epidermal cells, and after 5 days of culture, use methylene blue (SIGMA). Stained. The number of colonies was determined by visual inspection of the stained ones. The results are shown in Figure 3.

The growth rate was measured by a cell count method and MTT assay. The cell number was directly measured using a hemocytometer overnight. MTT Atsee utilizes the color change of MTT (3- (4,5-dimethylthiozol-2-yl) -2,5-diphenyltetrazoliumbutamate) (manufactured by SIGMA). Is the way. MTT is a yellowish compound that is degraded by the active mitochondria of living cells to produce a dark blue formazan compound. For this reason, only living cells were stained with this dye, and the relative cell amount was simply measured. The results are shown in FIGS. 4 and 5, respectively.

The colony forming ability indicates the growth potential of the cell. As shown in FIG. 3, the colony forming ability of the limbal epithelial cells was about twice that of the epidermal cells, and was significantly higher than that of the oral epithelial cells. In addition, it was shown that the proliferation rate of inner epithelial cells was faster than that of epidermal cells and oral epithelial cells (see FIGS. 4 and 5). BH ,, _n

Based on these results, lining epithelial cells have the characteristic of having a high proliferative capacity, and are a distinctly different cell group from oral epithelial cells, and can form sheets more rapidly when forming cell sheets. That is, it is suggested that the sheet forming ability is high.

(2) Thigh

Next, the antigenicity of the oral mucosal epithelial cell sheet was examined. Antigenicity was determined by assay of the expression of IA and Thy-1 antigens on both limbal and oral epithelial cells. Atsey's method is as follows.

In accordance with the method described above, limbal epithelial cells and epidermal cells were obtained from 5-6 week old male mice (BALB / c, IA = d, Nippon S.L.C., Hamamatsu). Similarly, oral epithelial cells and epidermal cells were obtained from male mice of the same age (C3H / Hen, IA = k). Anti-IA ^d antibody (Ce clarlane) was applied to the isolated BALB / c mouse cells, and anti-IA ^k antibody (Becton Dickins on) was applied to C 3 H / Hen mouse cells. After incubation at 4 ° C for 60 minutes, rabbit complement was added, followed by incubation at 37 ° C for 60 minutes. Before the start of the culture, on the third, fifth and seventh days of the culture, the cells are collected, and FITC-conjugated anti-MHC class II antibody (Becton Dickinson) is added at a concentration of 1/300, and the mixture is incubated at 4 ° C for 45 minutes. After that, the cells were observed with a fluorescence microscope. Next, the numbers of IA antigen-positive cells and Thy-1 antigen-positive cells (dendritic cells) were measured using flow cytometry. Figure 6 shows the results.

As shown in FIG. 6, the expression of Thy-1 antigen and IA antigen in the limbal epithelial cells both decreased with the lapse of the culture period. This IAfc¾ is considered to be expressed on immunocompetent cells, Langerhans cells, in limbal epithelial cells. In the limbal epithelial cells, the expression of both cell antigens ΙΑί¾ and Thy-1 共に declined with time, suggesting that the immune response became weaker with the passage of the culture period.

On the other hand, Thy-1 antigen expression in oral epithelial cells was different from IA antigen expression, and was shown not to decrease even after the culture period. For this reason, oral epithelial cells did not show a decrease in the amount of cellular antigens compared to inner epithelial cells, and after the culture period This suggests that the immune response is not reduced as much as in the limbal epithelial cells.

These facts suggest that limbal epithelial cells are a group of cells different from oral epithelial cells due to their low aggressiveness and can form cell sheets that are unlikely to undergo rejection even when transplanted. .

III. Formation of mucosal epithelial cell sheet

(1) Preparation of feeder cells

3Τ3-J2 cells as feeder cells were treated with 4 g / ml mitomycin C (Kyowa Ugly, Tokyo) in serum-free DMEM. After 2 hours, PBS (-) was rinsed several times to remove mitomycin C, after trypsinization, so that it can be seeded at a density of 1 X 10 ⁴ cells / cm ^2, to prepare a cell suspension.

(2) Sheet formation

1 and suspensions adjusted oral inner mucosal epithelial cells as X 10 may seeded ⁴ / cm ² in the cell density was adjusted to allow seeding at a cell density of 1 X 10 ⁴ / cm ² supported The cell suspension was added to a 35 mm dish in an amount of 1 ml, and cultured in an EFM medium. Every 2-3 days, the medium was replaced with fresh EFM selective medium. Oral mucosal epithelial cells seeded and cultured on supporting cells can be used as a mucosal cell sheet that can be used by culturing on supporting cells for about 10 days (three to five layers, about 100Π1) Became. On the other hand, since the supporting cells have lost their proliferative ability, they died during the culture period of the oral mucosal epithelial cells, and were removed from the culture system when the mucosal epithelial cell sheet was formed. The formed mucosal epithelial cell sheet is composed of oral and inner lining mucosal epithelial cells. When using this mucosal epithelial cell sheet, the epithelial cell sheet is detached from the dish with dispase (400 PU / ml). Washed twice with PBS.

IV. Properties of mucosal epithelial cell sheet

(1) Ability to form mucosal epithelial cell sheet

The mucosal epithelial cell sheet of limbal epithelial cells formed as described above was able to be used as a graft after culturing for about 10 days. On the other hand, the oral epithelial cell sheet requires about 2 weeks of culture time. Therefore, the lining epithelial cell sheet is a graft that can be used earlier than the oral epithelial cell sheet. Rukoto has been shown.

(2) Morphological characteristics of mucosal epithelial cell sheet

Next, in order to examine the morphological differences in the cell sheet between the lining epithelial cells and the oral epithelial cells, the cells were observed with an electron microscope. Inner marginal epithelial cell sheet, oral epithelial cell sheet, and epidermal cell sheet were each formed as described above, fixed with a Kernovski fixative for 2 hours, sliced, and photographed with a transmission electron microscope. . The results are shown in FIGS. 7A, 7B and 7C.

As shown in FIG. 7A, the mucosal epithelial cell sheet composed of the limbal epithelial cells has a clearly different morphology from the epidermal cell sheet (FIG. 7C). In addition, compared with the mucosal epithelial cell sheet composed of oral epidermal cells (FIG. 7B), it was shown that the mucosal epithelial cell sheet composed of the inner peripheral epithelial cells had a wider cell gap.

Such a wide intercellular space was a characteristic property of the limbal epithelial cells, and was shown to be retained in the mucosal epithelial cell sheet composed of the limbal epithelial cells. It was also confirmed that a mucosal epithelial cell sheet composed of limbal epithelial cells could be identified based on such morphological characteristics.

(3) Antigenicity of mucosal epithelial cell sheets

In order to examine the antigenicity of the mucosal epithelial cell sheet, engraftment was performed using mouse cells. By the same method as described above, lining epithelial cells, oral epithelial cells, and epidermal cells were collected and layered by culturing for a predetermined period. Once the cells were stratified, they were detached with dispase (400 PU / ml) and washed twice with PBS to prepare individual cell sheets as transplants. The cell sheets were collected from a nude mouse (5-6 weeks old, male, BALB / cnu / nu) under general anesthesia by intraperitoneal administration of Bentbarbi (0.04 mg / g) (Japan (S.L.C., Hamamatsu).

The method of transplantation was the method of Barrandon et al. (Barrandon et al., J. Invest. Dermatol., (1998) 91, 315-318) and the method of Sugimura et al. (Sugimura et al., J. cranio-maxillofac Surg., (1997) 24 , 35 2-359).

That is, after thoroughly disinfecting the back of the mouse with 10% A x30 mm rectangular flap was made to expose the muscular layer. A sterile silicone membrane was placed over the entire exposed mouse muscle layer to isolate the flap from the muscle layer. Next, each of the prepared cell sheets was placed on a sterilized silicone membrane as a carrier for transplantation, and transplanted onto the silicone membrane so that the cells of the sheet were in contact with the inside of the flap. Then, the flap was returned so as to cover the transplanted mucosal epithelial cell sheet and silicone membrane, and the graft was terminated by suturing with a 5-Nail thread.

From the third day of transplantation, the flap was raised again every three days, and each cell sheet transplant was visually observed. Cells that were whitened and detached were judged to be dead because they could not survive. The engraftment rate was calculated from the ratio of the area of the engrafted cells to the transplanted surface. Figure 8 shows the results.

As shown in Fig. 8, the inner marginal mucosal epithelial cell sheet (mouth) slightly reduced the engraftment rate in about 2 weeks, and maintained the engraftment rate after 3 weeks. In contrast, almost all cells of the epidermal cell sheet (△) died about 10 days after transplantation. In addition, the cell sheet (garden) of the oral epithelial cells began to decrease in survival rate after about 12 days, and all died after about 2 weeks. Killing of the epidermal and oral epithelial cell grafts is thought to be due to rejection.

It is thought that the maintenance of a high cell sheet engraftment rate of inner mucosal epithelial cells is related to a decrease in the expression of Thy-1 antigen in inner mucosal epithelial cells (see Fig. 6). As described above, since the mucosal epithelial cell sheet of the present invention is formed from the oral mucosal epithelial cells, the mucosal epithelial cell sheet can be prepared from the cells in a short time, and the obtained mucosal epithelial cell sheet is obtained. The cell sheet can be used as a graft for a long time.

In addition, since the cell sheet is formed from the oral mucosal epithelial cells adhered to the extracted tooth, the oral mucosal epithelial cells conventionally discarded as biological waste can be effectively used.

Furthermore, the gene is transfected into the oral mucosal epithelial cell constituting the present mucosal epithelial cell sheet by ordinary means in the same manner as other mucosal cells, and the oral mucosal epithelial cell sheet into which the desired target gene has been introduced is obtained. Obtainable. Such a gene-introduced oral inner marginal mucosal epithelial cell sheet has various functions depending on the introduced target gene. Can be fulfilled. Industrial applicability

The mucosal epithelial cell sheet of the present invention can be prepared more quickly and can be used as an implant that can be engrafted for a long time.

In addition, various genes can be introduced into the oral mucosal epithelial epithelial cells to form a sheet, so select the target gene, such as a graft with high engraftment efficiency and a carrier for gene therapy. Thus, it can be used as an implant having a desired function.

By the method for producing a mucosal epithelial cell sheet of the present invention, a transplant that can be engrafted for a long period can be prepared more quickly. Further, it is possible to provide a useful method of use for the oral marginal mucosa Ji giant epithelial cells which have been discarded without any special use. The description in this specification is merely for the purpose of describing the present invention and should not be treated as a limitation of the present invention, and various changes may be made without departing from the spirit and scope of the present invention. .

Claims

The scope of the claims

1. A mucosal epithelial cell sheet composed of multiple layers of oral mucosal epithelial cells forming a part of the gingival sulcus, which are continuously attached to the oral mucosal epithelial cells and form a part of the gingival sulcus.

2. The mucosal epithelial cell sheet according to claim 1, wherein said oral mucosal epithelial cells are derived from those adhered to teeth and separated from gingiva during tooth extraction.

3. A method for producing a mucosal epithelial cell sheet comprising a plurality of layers of oral mucosal epithelial cells forming a part of the gingival sulcus, which are continuously attached to the oral mucosal epithelial cells and form a part of the gingival sulcus. hand,

Obtaining the oral mucosal epithelial cells and supporting cells that support the growth of the oral mucosal epithelial cells,

A mucosal epithelial cell sheet formed by culturing the oral mucosal epithelial cells in the presence of the supporting cells to form a mucosal epithelial cell sheet composed of a plurality of layers of the oral mucosal epithelial cells; Get

A method for producing a mucosal epithelial cell sheet comprising:

4. The method for producing a mucosal epithelial cell sheet according to claim 3, wherein the oral inner marginal mucosal epithelial cells are separated from the gingiva while being attached to the teeth during tooth extraction. .

5. The method according to claim 3, wherein the method further comprises, prior to the simultaneous culture of the supporting cells and the oral mucosal epithelial cells, eliminating the dividing ability of the supporting cells by irradiation with a drug or radiation. A method for producing the mucosal epithelial cell sheet according to the above.