JP2002331025A - Cornea curing cultured epitheliocyte sheet and its manufacturing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cornea curing cultured epitheliocyte sheet which is effective to cure a cornea and which can be manufactured easily. SOLUTION: A method for manufacturing the cornea curing cultured epitheliocyte sheet comprises the steps of adopting an epitheliocyte originated from a mouth mucosa cell, curing the epitheliocyte on a supporting cell, and manufacturing the cornea curing cultured epitheliocyte sheet having at least one layer of the cured epitheliocyte and utilized for a corneal epithelial disease.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cultured epithelial cell sheet for corneal treatment used for corneal treatment of a patient's eye and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, eye drops and ointments have been used for inflammation such as keratitis and conjunctivitis, and corneal diseases such as corneal epithelial damage. This treatment with only eye drops or ointment is applied to relatively mild corneal diseases.

[0003] In contrast, in moderate to severe corneal diseases, for example, keratoconjunctivitis sclera, the subepithelial scar tissue of the corneal conjunctiva is removed and then the amniotic tissue is exposed on the exposed sclera or corneal stroma. Treatment is known. Diseases to be treated by a similar operation method include Stevens-Johnson syndrome, ocular pemphigus, chemical / burn, and pterygium.

In recent years, a technique for culturing corneal epithelial cells has been established. It is also conceivable that cultured corneal epithelial cells produced by this technique are used for treating the above diseases.

[0005]

[0005] However, in the treatment by amniotic tissue encroachment, not only is it difficult to obtain the amniotic tissue but also it is difficult to secure a sufficient amount of the tissue, and furthermore, it is difficult to obtain an immune rejection reaction. There is a problem that needs to be considered.

On the other hand, in the treatment using cultured corneal epithelial cells, the amount of corneal epithelial cells that can be collected is extremely small.
There is a problem that it is difficult to secure a sufficient amount of organizations. Also, regarding the culture of corneal epithelial cells, it was also difficult to obtain a large number of cells by culturing because the number of base cells was small.

SUMMARY OF THE INVENTION In view of the above problems of the prior art, it is an object of the present invention to provide a cultured epithelial cell sheet for corneal treatment which is effective for corneal treatment and can be easily prepared, and a method for producing the same.

[0008]

The cultured epithelial cell sheet for corneal treatment of the present invention has a layer of cultured epithelial cells derived from epithelial cells other than corneal epithelial cells and is used for corneal epithelial diseases. It is characterized by: Since such a cultured epithelial cell sheet has a layer of epithelial cells other than corneal epithelial cells, when transplanted to the cornea for the purpose of treating a corneal epithelial disease, a physiologically active substance is obtained from the cultured epithelial cells. Produced for surrounding corneal epithelial cells. Thereby, regeneration of corneal epithelial cells is promoted, and efficient corneal treatment can be performed.

In another aspect of the present invention, the cultured epithelial cell sheet is characterized in that the cultured epithelial cells are derived from oral mucosal cells. Since such a cultured epithelial cell sheet is composed of epithelial cells derived from oral mucosal epithelial cells, the characteristic of oral mucosal epithelial cells, which is low in immunogenicity, can be maintained for the cultured epithelial cell sheet. As a result, a cultured epithelial cell sheet having low immunogenicity can be obtained, and rejection can be suppressed.

The method for producing a cultured epithelial cell sheet for corneal treatment according to the present invention comprises the steps of: disseminating epithelial cells other than corneal epithelial cells on or together with the supporting cells; And a culturing step of culturing in a sheet form. According to this method, the epithelial cells other than the corneal cells are cultured together with the supporting cells, so that the stratified epithelial cell sheet can be easily produced under the influence of the product produced by the supporting cells. Moreover, the stratification of epithelial cells is easily performed by the presence of the supporting cells, and a sufficient amount of a physiologically active substance can be produced. Thereby, an epithelial cell sheet effective for corneal treatment can be efficiently produced.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The cultured epithelial cell sheet for corneal treatment of the present invention is a sheet-shaped cultured epithelial cell sheet for corneal treatment having at least one layer of cultured epithelial cells derived from epithelial cells other than corneal epithelial cells. . Since such a cultured epithelial cell sheet for corneal treatment is composed of epithelial cells other than corneal epithelial cells, it is possible to ensure a sufficient number of cells compared to corneal epithelial cells, By culturing the sheet, it can be provided early and easily. Further, after transplantation into the cornea, a physiologically active substance such as EGF (epithelial cell growth factor) is released into the contacting cornea, so that regeneration of the corneal epithelial cells at the transplanted portion can be promoted, and thereby corneal damage And corneal disease can be treated efficiently. In addition, when epithelial cells are obtained from the patient himself (autologous), immune rejection can be avoided and can be integrated with corneal epithelial cells for a longer period of time. . Hereinafter, the main cultured epithelial cell sheet will be described.

As the epithelial cells constituting the cultured epithelial cell sheet of the present invention, epithelial cells other than corneal epithelial cells, that is, skin epidermal cells, oral mucosal epithelial cells, and other gastrointestinal epithelial cells can be used. It is not particularly limited as long as it is an epithelial cell. Among them, oral mucosal epithelial cells are particularly preferable because they can be easily collected and further have characteristics such as a higher cell proliferation rate and lower immunogenicity than, for example, epidermal cells. In particular, one of the oral mucosal epithelial cells,
Oral mucosal epithelial cells that form part of the gingival sulcus are more preferred because they have higher division ability and are less likely to cause rejection than ordinary oral mucosal epithelial cells.

[0013] These mucosal epithelial cells can be collected from a suitable organism. For example, the oral mucosa cells can be collected from the oral cavity of a healthy subject together with the submucosal tissue.
Since the collected mucosal tissue contains connective tissue in addition to mucosal cells, it is necessary to isolate mucosal cells. In the isolation of mucosal cells, an appropriate enzyme such as dispase or trypsin is used to remove connective tissue and dermis from the collected mucosal tissue. Also, debris and the like are removed using a nylon mesh or the like.

In addition, skin epidermal cells, oral mucosal epithelial cells, and the like can be directly collected from the patient who performs corneal treatment. Epithelial cells collected directly from such patients themselves can avoid immune rejection,
preferable.

The cultured epithelial cell sheet of the present invention has at least one epithelial cell layer as described above. The epithelial cell layer may have at least one layer. However, it is preferable that the epithelial cell layer has a multilayer structure in which two or more layers are stacked from the viewpoint of imparting appropriate strength at the time of transplanting the cultured cell sheet. In addition, the cultured epithelial cell sheet may have a layer other than the epithelial cell layer that does not affect the cultured epithelial cell sheet at all, but those containing other types of layers require time and effort to prepare, so that they can be efficiently produced. From the viewpoint of producing a cultured epithelial cell sheet, it is preferable to include only the cultured epithelial cell layer.

Next, a method for producing the cultured epithelial cell sheet for corneal treatment of the present invention will be described. The cultured epithelial cell sheet for corneal treatment of the present invention is produced by a method including a seeding step of seeding the above-mentioned epithelial cells on or along with the supporting cells, and a culturing step of culturing the seeded epithelial cells in a sheet form. Is done.

In the present invention, feeder cells are used to grow mucosal epithelial cells, which are transfected cells, in a layered manner with high efficiency. The supporting cells include, for example, fibroblasts, for example, mouse NIH3T3 cells, 3T3J2, and Swiss 3T3, and 3T3J2 cells are preferable from the viewpoint of the thickness of the obtained transfected cell layer and the growth rate of the transfected cells on the supporting cells. . The feeder cells lose their proliferative ability so as not to interfere with the growth of the introduced cells. The proliferation ability can be eliminated by a method well known in the art, for example, treatment with mitomycin C or irradiation. In addition, culture
This is performed using a culture vessel such as a normal plastic dish.

For epithelial cells, feeder cells are seeded first, and
Seeding after attaching to the bottom of the
May be sowed together. At this time, the epithelial cells were 1 × 1
0^ThreePieces / cm^TwoAbove, preferably 1 × 10^FourPieces / cm^Two~ 1
× 10^FivePieces / cm^TwoCan be seeded at a cell density of
On the other hand, the feeder cells were 1 × 10^ThreePieces / cm^Two~ 1 × 10 ^FourPieces
/ Cm^ThreeThe seeding can be performed as described above. The supporting cells are
If it is less than this, it plays a sufficient role as a supporting cell
In excess of this, and more
It is not preferable because it hinders. In addition, epithelial cells
If it is less or more than the range, it can grow efficiently
It is not preferable because it cannot be done.

In the present invention, a growth medium is used to grow epithelial cells into sheets in the presence of 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, epithelial cells readily form layers and become sheet-like. On the other hand, the feeder cells die only by supporting the growth of the epithelial cells, and when the epithelial cells form a sheet, they no longer exist in the culture system.

It is preferable to use an epithelial sheet forming medium (EFM) having a sheet forming ability as the growth medium. EF
M is a medium that is usually used to grow epidermal cells in layers, and is composed of a 3: 1 mixture of DMEM and Ham F-12 medium, 5% FBS, 5 μg / ml insulin, 5 μg / Ml transferrin, 2 ×
10 ⁻⁹ M triiodothyronine, 1 × 10 ⁻⁹ M cholera toxin, 0.4 μg / ml hydrocortisone, 10
0 U / ml penicillin, 0.1 μg / ml kanamycin, 0.25 μg / ml amphoteritin B and 10
ng / ml of human recombinant epidermal growth factor (E
GF) and the like.

The sheet-shaped cultured epithelial cells are separated from the culture vessel while maintaining the sheet shape. This separation can be performed by a method known in the art, and can be easily performed by using a suitable enzyme such as dispase to inhibit the adhesion of the basal layer.

The cultured epithelial cell sheet of the present invention is used for corneal treatment. Corneal disorders and diseases suitable for use of the cultured epithelial cell sheet of the present invention include scar keratoconjunctivitis, Stevens-
It is a corneal epithelial disease such as Johnson syndrome, ophthalmic pemphigus, chemical / burn, pterygium. When the cultured epithelial cell sheet of the present invention is used for such corneal disorders and diseases, the cultured epithelial cell sheet is transplanted to a corneal defect. The transplant is
What is necessary is just to cover the cultured epithelial cell sheet so as to cover the entire surface of the defective portion. From the viewpoint of protection of the transplanted portion and prevention of drying, it is preferable to cover the transplanted portion with a contact lens or the like.

The cultured epithelial cell sheet of the present invention comes into contact with a corneal defect after transplantation for corneal treatment. At this time, the cultured epithelial cell sheet can release a physiologically active substance to the defective part and promote the cornea epithelization, so that the cornea can be treated by efficiently regenerating the cornea. The cultured epithelial cell sheet only needs to be present at a time necessary for regeneration of the cornea to be treated, and may then fall off.

[0024]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described with reference to the drawings, using an oral mucosal epithelial cell as a cultured cell as an example. However, the present invention is not limited only to the following examples.

The oral mucosal epithelial cells to be seeded cells were collected from healthy oral mucosa with the patient's consent. The collected oral mucosal tissue was cut into small pieces after removing the submucosal tissue with scissors. Small pieces of mucosal tissue were treated with phosphate buffer (PBS) containing 1000 U / ml penicillin, 1 mg / ml kanamycin and 2.5 μg amphotericin B,
It was immersed twice at 37 ° C. for 30 minutes. Then, 1000PU
/ Ml of dispase (manufactured by Godo Shusei Co., Ltd.) in Dulbecco's modified Eagle's medium (DMEM) for 16 hours at 4 ° C., then 0.2 minutes at room temperature for 30 minutes.
Cells were separated by enzyme treatment with 5% trypsin. Enzyme activity was measured in DME containing 10% fetal bovine serum (FBS).
Stopped by washing with M. Then, 10% F
Stir for 30 minutes in DMEM containing BS, then add
Oral mucosal epithelial cells were isolated by filtering the debris with a μm nylon gauze. The cell suspension was adjusted to a cell density of 1 × 10 ⁴ cells / ml.

3T3-J2 cells, selected as feeder cells, were treated with 4 μg / ml mitomycin C in serum-free DMEM and inactivated. Two hours later, mitomycin C was removed by washing (rinsing) several times with PBS (-), and the cells were treated with trypsin to prepare a cell suspension of 1 × 10 ⁴ cells / ml.

[0027] 1 × 10 ⁴ and a suspension of oral mucosa epithelial cells was adjusted to a cell density of cells / ml, 1 × 10 ⁴ cells / ml
The suspension was added to a 35 mm dish in an amount of 1 ml, and cultured in an EFM medium (epithelial sheet forming medium). The medium was changed every 2-3 days with fresh EFM selection medium. When the epithelial cells were seeded and cultured for about 10 days, a usable mucosal epithelial cell sheet was formed. On the other hand, since the proliferative ability of the supporting cells has been lost, the supporting cells have died during the culture period of the oral mucosal epithelial cells, and have been removed from the culture system when the mucosal epithelial cell sheet is formed.

The cultured oral mucosal epithelial cell sheet is cut, stained with a hematoxylin-eosin stain,
A stained cross-sectional image was obtained. Observation of this stained cross-sectional image revealed that the formed mucosal epithelial cell sheet was composed of oral mucosal epithelial cells. When using this mucosal epithelial cell sheet, the epithelial cell sheet is dispase (400 PU / m
Peel off the dish in l) and wash several times with PBS before using.

The following transplantation was carried out for the purpose of transplanting the cultured oral mucosal epithelial cell sheet prepared by the above-mentioned method into a corneal defect layer. Corneal chemical wounds were made on 5-year-old male Japanese white rabbit corneas using sodium hydroxide (see FIG. 1 (a)). That is, a 0.1 N sodium hydroxide solution was immersed in a filter paper having a diameter of 8 mm, and the filter was brought into contact with the rabbit cornea for 90 seconds to damage the cornea. Thereafter, it was confirmed that an epithelial defect having a diameter of 8 mm or more was present on the corneal surface using a flowless staining solution and black light (see FIG. 1 (c)).

In the same part, commercially available soft contact lenses were prepared by using a previously prepared cultured oral mucosal epithelial cell sheet using human oral mucosa and a cultured oral mucosal epithelial cell sheet using the rabbit oral mucosal cells (autologous cells). As carriers (see FIG. 1 (b)), each of them is transplanted (covered with a cultured oral mucosal epithelial cell sheet so as to cover the entire epithelial defect) (see FIG. 1 (d)), and protection of the transplanted part is achieved with a disposable contact lens. went. Thereafter, the upper and lower eyelids of the transplanted eye are placed 3-0 so that the contact lenses used do not fall off.
The suture was sutured with a silk thread to complete the transplant. After transplantation, injection of antibiotics was administered for the purpose of preventing wound infection.

On day 5 after the transplantation, the upper and lower eyelids fixed by suture were released, and the transplanted portion was observed. The transplanted oral mucosal epithelial sheet could be observed on the corneal surface according to the transplantation rule as an irregular surface with macroscopically unevenness (see FIG. 2 (c)). On the other hand, these findings were not found in the non-transplanted group (see FIG. 2 (b)). FIG. 2A shows an observed image of a normal cornea as a comparative object. In addition, both were stained with Flores' staining solution and the degree of epithelialization was observed.As a result, there was almost no staining site on the transplanted side, and the experimentally created epithelial defect site was epithelialized from the surrounding or cultured oral mucosa. It was a finding that the epithelial sheet had survived. On the other hand, the non-transplant group showed positive flowless staining, and epithelialization from the surroundings was observed, but a clear epithelial defect was observed. This finding showed little difference between human oral mucosal cells and the rabbit oral mucosal cells (autologous).

Further, FIG. 3 shows a cross-sectional image of a stain with a hematoxylin-eosin stain. It can be observed that the corneal epithelial layer on the transplant side shown in FIG. 3 (c) has a sufficient thickness, as can be seen from comparison with the stained cross-sectional image of the normal cornea shown in FIG. 3 (a). . In contrast, it can be observed that the corneal epithelial layer on the non-implanted side shown in FIG. 3B remains thin.

On the seventh day after transplantation, the area occupied by the transplanted oral mucosal epithelial epithelial sheet on the cornea was reduced, suggesting that the oral mucosal cells were promptly replaced by smooth corneal epithelial cells. . On the 10th day of transplantation, no surface irregularities derived from the transplanted cultured oral mucosa were observed, and all the transplanted portions were replaced with corneal-derived cells. On the other hand, in the non-transplanted group, a corneal defect was observed punctate on day 10 after transplantation, and corneal epithelialization was delayed.

On day 7 of the transplantation, the transplanted portion was stained with hematoxylin-eosin and observed histologically. As a result, at the boundary between the transplanted cultured oral mucosal epithelial sheet and the corneal epithelium around the transplanted area, an image is seen in which the host corneal cells are progressing toward the center in the direction to remove the cultured oral mucosal epithelial sheet. It was suggested that corneal cells grew in the same area. The above findings were similar for both human oral mucosa-derived cells and autologous cells. These findings were not observed in the non-transplant group.

As described above, the cultured epithelial cell sheet of the present embodiment can be easily prepared because the oral mucosal epithelial cells serving as a culture base can be easily collected.

The cultured epithelial cell sheet comes into contact with the patient's cornea after surgery and releases various physiologically active substances (particularly various cell growth factors) from the cell layer. Promotes healing ability. In addition, when autologous tissue is used, immune rejection after transplantation is not induced, and it is considered that the method works advantageously in the subsequent corneal treatment.

[0037]

As described above, according to the present invention, it is possible to provide a cultured epithelial cell sheet for corneal treatment which is effective for treating the cornea and can be easily prepared.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a state of transplantation in an embodiment of the present invention, wherein (a) shows a rabbit cornea being treated with sodium hydroxide, and (b) shows a cultured oral mucosa using a soft contact lens as a carrier. It is a figure which shows the epithelial cell sheet, (c) which shows the corneal epithelium defect before transplantation, and (d) which shows a mode that the cultured oral mucosal epithelial cell sheet is transplanted.

2A is an observation image of normal corneal tissue, FIG. 2B is an observation image of non-transplanted corneal tissue, and FIG. 2C is an observation image of corneal tissue 5 days after transplantation.

FIG. 3 is a diagram showing cross-sectional images of (a) a normal corneal tissue, (b) a corneal tissue on the non-implanted side, and (c) a corneal tissue on the implanted side 5 days after transplantation, stained with a hematoxylin-eosin stain. is there.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kenichiro Hatake 2-10-3 Itakuracho, Kariya-shi, Aichi 102 Sun Village Itakura 102 (72) Inventor Koji Hirano 2-1804-1 Kifune, Meito-ku, Nagoya-shi, Aichi F-term (Reference) 4B065 AA93X BC41 CA44 4C081 AB21 BA12 CD34 DA02 EA02

Claims (3)

[Claims] [Claim 1] A cultured epithelial cell sheet for corneal treatment, comprising at least one layer of cultured epithelial cells derived from epithelial cells other than corneal epithelial cells, and used for corneal epithelial diseases. 2. The cultured epithelial cell sheet for treating cornea according to claim 1, wherein the cultured epithelial cells are derived from oral mucosal cells. 3. A seeding step of seeding epithelial cells other than corneal epithelial cells on or together with the supporting cells, and a culturing step of culturing the cells in a sheet in the presence of the supporting cells. A method for producing a cultured epithelial cell sheet for treating cornea, which comprises the steps of: