JPH10277143A - Transplant and manufacture of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve the take rate in the transplantation, reproduce the skin or the like, and permanently close a wound, by embedding the fibroblast derived from an epithelium tissue, in a sheet of agglutination human plasma proteins or agglutination human fibrin, and attaching squamous epithelium cells to a surface of the sheet. SOLUTION: The fibroblast exists in a condition that it is dispersed in a gel sheet made of fibrin or agglutination human plasma proteins, and a surface of the sheet is covered with the squamous epithelium cells, to obtain the transplantation piece. That is, the dermis fibroblast is suspended in a solution containing fibrinogen or the like, and is expanded in an incubator. Then thrombin and calcium are added to the same, to gglutinate fibrinogen, whereby the fibrin sheet in which the fibroblast is embedded, can be obtained. Then the low calcium serum-free medium or the like is added to the incubator, is left as it is for several hours, and the moisture is substituted for the culture medium. Then the human epidermis cell is sowed on the surface, to attach the cell. Whereby the transplantation piece of a high take property, can be obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a graft, and more particularly to a graft used for treating a full-thickness wound of human skin or other squamous epithelial tissues and a method for producing the same.

[0002]

2. Description of the Related Art Severe burns, surgical operations, and the like often result in extensive skin or other squamous tissue full-thickness wounds. Since these wounds have lost all the components of the tissue, natural regeneration and healing cannot be expected as in the case of a wound that does not reach the full thickness, and transplantation of the tissue is necessary for treatment. For such tissue transplantation, for example, epidermal cells and fibroblasts, which are constituent cells of the skin, can be expanded and cultured 10 ³ to 10 ⁶ times in vitro. A treatment method has been reported in which the tissue is collected, grown and cultured, and then transplanted to a wound.

[0003] Transplantation methods used for these treatments are broadly divided into a single epidermal cell transplantation method in which only epidermal cells grown and cultured are transplanted and a dermal substitute made of collagen gel containing fibroblasts. A “composite artificial skin transplantation method” for transplanting a skin-like structure containing both fibroblasts and epidermal cells produced by growing and growing epidermal cells is known.

[0004] The epidermal cell transplantation method includes a method in which only epidermal cells are proliferated and cultured, and the resulting epidermal cells are layered to form a sheet of epidermal cells and transplanted. There is a method of transplanting cells in an isolated state.
No. 8863 discloses keratinocytes (epidermal cells).
A method of growing and growing epidermal cells mixed with 3T3 cells irradiated so as not to proliferate has been disclosed. Japanese Patent Publication No. 23231/1990 discloses epidermal cells on a culture vessel obtained in this manner. A method of preparing a sheet for transplantation by treating a culture with an enzyme is disclosed in Japanese Patent Application Laid-Open No. 02-174.
No. 848 discloses a method for culturing epidermal cells together with EGF in a collagen-coated culture vessel, and Japanese Patent Application Laid-Open No. 04-45785 discloses a method for culturing epidermal cells using aggregated human plasma protein as a support. It has been disclosed.

[0005] On the other hand, in the composite artificial skin transplantation method, fibroblasts cultured in a collagen gel or collagen sponge are dispersed, and epidermal cells are cultured and layered on these surfaces to produce a structure similar to the skin. Transplantation method, for example, Japanese Patent Publication No. 61-33593, Japanese Patent Publication No.
53505, JP-A-04-129563, JP-A-06-
Many documents are known, including 292568.

[0006]

However, the above-described method for transplanting epidermal cells alone has low engraftability when directly transplanted into a full-thickness defect wound lacking the dermis component, and is difficult to engraft for a long period of time. Therefore, it was difficult to directly adapt to a wide range of full-thickness defect wounds. Therefore, for a full-thickness defect wound, a composite artificial skin transplantation method for transplanting a skin-like structure having a dermis-like structure in which fibroblasts are dispersed in collagen gel or the like is preferable. However, in this transplantation method, a substance such as collagen that disperses fibroblasts is transplanted into the subcutaneous epidermis, and only a low engraftment rate has been reported due to the immune response to the substance and susceptibility to infection. Did not. As described above, it was practically difficult to permanently close the wound by implanting a graft consisting of skin cells grown and cultured on the wound on the body surface where the entire skin layer was defective.

Accordingly, the present invention reproduces skin and other squamous epithelial tissues with an excellent engraftment rate when transplanted into a wound on the body surface such as a wound in which the entire skin layer is defective or a squamous epithelial tissue wound in a living body. It is another object of the present invention to provide an implant capable of permanently closing a wound and a method for producing the implant.

[0008]

Means for Solving the Problems The present inventors transplant isolated human skin or other cells of squamous epithelial tissue into a full-thickness wound on the surface of the body, and obtain a large area of human skin or other cells. As a result of intensive studies on the method of reproducing squamous epithelial tissue, human epidermis cells or other squamous epithelial cells grown and cultured were cultured on the surface of aggregated human plasma protein or aggregated human fibrin in which human dermis or fibroblasts derived from the organ were embedded. The present inventors have found that the use of the attached graft makes it possible to reproduce the skin or other squamous epithelium having a large area on the surface of the living body and to permanently close the wound on the body surface, thereby completing the present invention.

That is, in the graft of the present invention, fibroblasts derived from epithelial tissue are embedded in a sheet of aggregated human plasma protein or aggregated human fibrin, and the squamous epithelial cells derived from the epithelial tissue are coated on the surface of the sheet. Wherein fibroblasts derived from human dermal tissue are embedded in a sheet of aggregated human plasma protein or aggregated human fibrin, and epidermal cells are attached on the sheet surface. It is characterized by.

[0010] In addition, the method for preparing a graft of the present invention comprises isolating squamous epithelial cells and fibroblasts from epithelial tissue, and squamous epithelial cells and squamous epithelial cells derived from the same tissue as the isolated epithelial tissue. Preparing fibroblasts, respectively, culturing the obtained squamous epithelial cells and the fibroblasts, and enclosing the cultured fibroblasts in aggregated human plasma protein or aggregated human fibrin. Embedding and comprising the steps of attaching squamous epithelial cells to the surface of aggregated human plasma protein or aggregated human fibrin embedded with the obtained fibroblasts, and further, epidermal cells from skin tissue and Isolating fibroblasts and preparing epidermal cells and dermal-derived fibroblasts, respectively, and expanding and culturing the obtained epidermal cells and fibroblasts, respectively. Embedding fibroblasts grown and cultured in aggregated human plasma protein or aggregated human fibrin, and attaching epidermal cells to the surface of aggregated human plasma protein or aggregated human fibrin in which the obtained fibroblasts are embedded The method is characterized by comprising the steps of:

The graft and the method for producing the same according to the present invention can be applied to all mammals. explain.

The epithelial tissues to be used in the present invention are all tissues containing squamous epithelial cells, and include all normal tissues. Examples of these tissues include, for example, skin and oral mucosa. , Esophagus, tongue and the like. In addition, the human cells used in the present invention may be modified from those obtained from normal tissues as well as cells derived from normal tissues by introducing foreign genes or treating with drugs in vitro (in vitro). Strained cells can also be used.

The squamous epithelial cells used in the present invention are collected from normal epithelial tissue of the organ to be transplanted. For example, when performing skin transplantation, human epidermal cells can be obtained by the following method. That is, the human stratum corneum or full thickness skin is aseptically collected and washed with sterile physiological saline. Use dispase II (2,000 U / ml) for 37
The human epidermal layer is separated by treating at 20 ° C. for 20 to 40 minutes and washed with physiological saline. 0.25% more
The mixture is treated with trypsin at about 37 ° C. for 5 minutes, and stirred in a serum-containing medium to disperse epidermal cells. The dispersed epidermal cells were seeded in a culture vessel at a density of about 5 × 10 ⁴ / cm ² ,
About 0% FCS or MEM Alpha with human serum
And culture for 24 hours. Thereafter, the medium is replaced with a low calcium serum-free medium (MCDB153), and the culture is continued for about one week. Thereafter, the cultured epidermal cells are detached by trypsin treatment, subcultured, and grown and cultured using a low calcium serum-free medium (MCDB153) until a desired cell number is obtained. In the case of other squamous epithelial cells,
Squamous epithelial cells can be obtained by the same method. For example, oral mucosal tissues can be grown and cultured by the completely same method. The human epidermal cell culture described above can also be carried out by co-culturing with irradiated fibroblasts or fibroblast cell lines as described in JP-B-57-18863.

The fibroblast used in the present invention is a fibroblast derived from an epithelial tissue from which squamous epithelial cells have been obtained. This is necessary for the transplanted squamous epithelial cells to proliferate, form a normal tissue structure, and survive for a long time after transplantation, and is derived from a tissue different from the epithelial tissue from which the squamous epithelial cells were obtained. For fibroblasts, long-term engraftment after transplantation tends to be difficult. On the other hand, the obtained fibroblasts may be autologous or others, and autologous or allogeneic fibroblasts can be used as long as they are derived from the same epithelial tissue. Autologous cells are preferable in consideration of antigenicity, but even when allogeneic cells are used, the engraftment rate is improved,
There is no problem in reconstructing a normal tissue structure, and similar results can be obtained. Allogeneic fibroblasts have weak expression of HLA antigen,
It may be because they survive for a relatively long time,
Because the expression of HLA antigens may become stronger due to inflammation, etc., and if a patient has already been sensitized to an allogeneic antigen or has an antibody, it may be rejected immediately and may not function Therefore, it is preferable to use autologous cells if possible.

As described above, fibroblasts are considered to give some information specific to the transplanted squamous cells in order for the transplanted squamous cells to differentiate and proliferate into normal tissues. It has not been possible to identify what factors the information is.

In the case of skin transplantation, human dermis-derived fibroblasts are used for human epidermal cells. The human dermis-derived fibroblasts can be obtained, for example, by the following method. The human split or full thickness skin is aseptically collected and washed with sterile physiological saline. Dispase II (2,000 U / ml) at 37 ° C, 20-40
The dermis layer is separated by fractionation and washed with physiological saline. After shredding, the cells are cultured for about one week in a MEM Alpha medium supplemented with about 20% FCS or human serum, and the proliferating fibroblasts are subcultured. In the same manner, other fibroblasts can be separated and grown and cultured from cells derived from tissues from which squamous epithelial cells have been collected, for example, submucosal tissues such as oral mucosa and esophagus can be grown in exactly the same manner. It can be cultured.

The aggregated human plasma protein or aggregated human fibrin used in the present invention supports or embeds squamous epithelial cells and fibroblasts, and is used for transplantation until the grafts survive and the skin tissue is reproduced. It is used as a substrate for maintaining the integrity of the pieces. Such a substrate can attach cells, is harmless to cells and living organisms,
Any substance having appropriate elasticity and water retention can be used. Examples of such substances include gelatin and collagen. However, the implant of the present invention
It is preferable that it does not show antigenicity after transplantation into a living body, and is decomposed and absorbed as quickly as possible. In this regard, aggregated human plasma protein or aggregated human fibrin is the most preferable substance. Furthermore, a sheet made of such aggregated human plasma protein or aggregated human fibrin can be easily handled as a graft, and can be fixed to a wound on the surface of a living body. On the side, fibroblasts are arranged neatly on the tissue side and show a histological image similar to normal epithelial tissue, while fibrin itself is rapidly decomposed and absorbed without causing inflammation, and complete epithelial tissue A favorable result is obtained in that reproduction can be achieved.

In addition, since fibrin can be prepared from the blood of a patient, a graft composed entirely of autologous organic components can be prepared by using it, if necessary. . At present, various methods of sterilizing fibrin have been reported, and there is no known risk of infectious disease. However, the use of autologous fibrin can completely prevent transmission of infectious diseases, including unknown ones.

The sheet-like aggregated human plasma protein or fibrin can be produced, for example, by the following method. Autologous blood is collected by adding sodium citrate (0.2%) or ACD solution (5% (V / V)) and 800 ×
Centrifuge at g for 20 minutes to obtain the supernatant (ie, plasma). This was placed in an incubator, and human thrombin (30 U /
ml) and calcium chloride (2 mM), and left at 37 ° C. for several hours to obtain an aggregated human plasma protein sheet.

Without using human plasma as it is, ethanol was added to the plasma, left at -4 ° C. overnight, centrifuged, and the precipitate was dissolved in physiological saline (so-called Cohn's first product). Plasma fraction containing fibrinogen, fibronectin, etc.)
Aggregated human plasma proteins prepared by the action of thrombin may be used. In this case, the fibrinogen concentration can be adjusted, and the hardness of the agglomerated sheet, the time required for decomposition after transplantation, and the like can be adjusted. Furthermore, using fibrinogen purified and sterilized by various methods such as salting-out method other than this, the first plasma fraction of Cohn or purified fibrinogen is once freeze-dried, and then dissolved in physiological saline at the time of use. The same thing can be obtained by using.

The concentration of fibrinogen in preparing a sheet of aggregated human plasma protein or fibrin is appropriately adjusted according to the size and thickness of the sheet to be prepared, the amount of fibrin in the gel constituting the sheet, and the like. Disassembly of the sheet,
From the viewpoint of absorbability, the decomposition rate of fibrin also depends on the concentration of fibrin in the sheet. Therefore, for use in transplantation, the fibrinogen concentration at the time of sheet preparation should be about 400 to 800 mg / dl. Preferably
When fibrinogen having such a concentration is used, by adjusting the thickness of the sheet to about 0.5 to 1 mm, a sheet having an appropriate hardness and a fibrin concentration can be obtained. The preferred results are also obtained from

The concentration of calcium added at the time of preparing these aggregated human plasma proteins or fibrin sheet is 2%.
mM or less is desirable in order to reduce the effect on cells, and when producing a fibrin sheet, aprotinin,
By adding a protease inhibitor such as urinastatin, the degradation of fibrin can be appropriately delayed.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The graft of the present invention exists in a state in which fibroblasts are dispersed in a gel-like sheet composed of fibrin or aggregated human plasma protein, and the surface of the sheet is a squamous cell. It is covered. Such an implant can be prepared by embedding fibroblasts from the tissue in fibrin or aggregated human plasma protein and attaching squamous cells to the surface. Embedding of fibroblasts can be performed by coexisting fibroblasts when fibrinogen is aggregated.Fibrinogen is aggregated so as to surround fibroblasts, and fibrin clots with embedded fibroblasts or It becomes a sheet.

For example, in the case of a skin graft for skin transplantation,
It is produced as follows. That is, dermal fibroblasts are suspended in a solution containing the above-mentioned fibrinogen and the like, and a fibrinogen solution in which dermal fibroblasts are suspended is prepared. Next, this solution is developed in an incubator or the like, and thrombin or calcium is added as described above to aggregate fibrinogen, thereby preparing an aggregated fibrin sheet in which fibroblasts are embedded. At this time, it is preferable that a small amount of a human albumin solution is dropped on the bottom surface of the incubator to cover the implant in order to facilitate peeling of the graft later. A low calcium serum-free medium (MCDB153) or the like is added to an incubator containing the aggregated fibrin sheet in which the fibroblasts are embedded, and 37
C for several hours to swell the aggregated fibrin sheet,
Replace the water contained in the fibrin clot with the medium.
Thereafter, the above human epidermal cells are seeded on the aggregated fibrin sheet, and cultured at 37 ° C. for about 3 to 24 hours until the cells adhere to the fibrin surface. The culture period can be extended to about one week, but when cultured for a long period of time, there is a tendency that the basal structure is destroyed and the graft survival rate is reduced. In order to prevent infection after transplantation, a medium containing an antibiotic may be used at the time of preparing the graft. However, in this case, an antibiotic having low cytotoxicity, for example, 500 μg / ml PIPC (piperacillin) or the like is used. Preferably, it is used.

The cell seeding density when seeding human epidermal cells is preferably about 1 to 4 × 10 ⁵ / cm ² , particularly 2 × 1 ⁵ / cm ^2.
About 0 ⁵ / cm ² is preferable in terms of the survival rate and in that the whole area of the implanted portion can be covered. Further, the ratio of epidermal cells to fibroblasts is preferably about 1: 2 to 2: 1 in terms of engraftment rate. In addition, 5 × 10 ⁴ / human epidermal cells
Seed at about ² cm ²
Similar results can be obtained even after about 8 hours of growth culture.

Next, the fibrin sheet to which the cells have been adhered by the culture is peeled from the incubator using tweezers or the like to obtain a transplant. The resulting grafts had approximately 1-2 epidermal cells on a fibrin sheet embedded with fibroblasts.
The cells are attached in a layer state, and each cell density has almost the same cell density several hours after seeding each cell as that at the time of seeding. In cells cultured for 24 to 48 hours after seeding, the cell density of each cell is higher than at the time of seeding, and the epidermal cells have a multilayer structure consisting of several layers. All of these grafts can be used as they are for transplantation, or they can be divided and used according to the shape of the transplant bed.

[0027] In addition, a graft can be prepared by a similar method using other squamous epithelial cells and fibroblasts. For example, an oral mucosal squamous epithelium can also be used as a fibroblast and cultured from a submucosal tissue by a similar method, and a graft can be prepared by a completely similar method.

In the present invention, the transplantation of the aggregated fibrin sheet containing the cells for transplantation and attached thereto, ie, the graft,
For example, it can be performed by the following method. With respect to the tissue defect to be transplanted, the above-mentioned graft is allowed to stand in such a direction that the surface on which the epidermis cells are not adhered faces the wound. If the graft is unstable, fibrin glue (eg, Beriplast (trade name,
It is also preferable to fix the implant by applying (eg, Boehringer) to the wound surface. A covering bandage is performed on the implant using a covering material having good moisturizing properties. Any material can be used as the coating material, as long as it has good moisturizing properties, is non-adhesive and has no cytotoxicity. Silicone membranes (for example, Tegaderm (trade name, manufactured by Sumitomo 3M), etc.) and petrolatum gauze can be used. preferable. At this time, it is effective to prevent infection by mixing vaseline with an antibiotic having no cytotoxicity, for example, 500 μg / ml of PIPC (piperacillin). Also, a breathable two-layer silicone film (for example, Biobrene (trade name, manufactured by Nippon Vilene Co., Ltd.), etc.) can be used. It is desirable to make it difficult. The fibrin sheet used as a substrate is usually decomposed and absorbed about 3 weeks after transplantation, disappears from the tissue, and the defective part is where the transplanted epidermal cells and fibroblasts proliferate to form new skin tissue. Coated.

[0029] The melting and disappearance of the fibrin sheet is due to proteolytic enzymes, and it is necessary to leave the sheet for a longer period to engraft the transplanted cells and completely repair the defective part. In order to suppress the action of these proteolytic enzymes, for example, aprotinin, or by adding a proteolytic enzyme inhibitor such as urinastatin, or by using a high concentration of fibrinogen during sheet production, the sheet disappears The period until can be adjusted to some extent.

The transplantation of the graft to which the cells for transplantation according to the present invention are carried out is not performed from the beginning to the tissue defect, but after the cells for transplantation are once cultured in the body, Can be adopted. This is suitable for transplantation of epithelial tissue other than the site where the transplantation operation is relatively easy, such as skin transplantation, and the transplantation result can be easily confirmed. For example, transplantation at the time of filling the oral mucosa with defects.
As a specific method, first, the skin of the living body to be transplanted is incised and peeled, and a pocket is made under the skin. An implant with cells for implantation is inserted into the pocket, and the incision is closed. In order to insert the graft, the cell may be inserted with the cell attachment surface facing the body surface or inside the body. Then
After 2 to 4 weeks, the skin is incised again, and the skin or other squamous epithelium consisting of the transplanted cells reproduced subcutaneously is cut off and transplanted to a desired site. In addition, when transplanting the graft in the subcutaneous pocket, if a silicone film or the like is placed on the surface where the epidermal cells are attached, deformation of the graft,
Preferred results are obtained in preventing the spread of cells.

In the above description, a sheet-shaped implant is described as an example. However, the shape of the implant is not limited to a sheet, and other shapes may be used as necessary. Can be. An implant having such a shape can be prepared by changing the amount of the fibrinogen solution when the fibrinogen solution is aggregated.

[0032]

Next, the present invention will be described in more detail with reference to experimental examples.

Experimental Example 1 1. Preparation of graft A skin cell was cultured from the skin obtained from the surgical specimen, and a graft (transplant graft) was prepared as follows.

The separated skin is aseptically collected and washed with sterile physiological saline. This is called Dispase II (2,000
After treatment at 37 ° C. for 20 to 40 minutes at 37 ° C. (U / ml), the epidermal layer is separated and washed with physiological saline. 0.25% more
The cells were treated with trypsin at 37 ° C. for 5 minutes and stirred in a serum-containing medium to disperse the epidermal cells. 5x dispersed epidermal cells
The cells are seeded in an incubator at a density of about 10 ⁴ / cm ² , and cultured for 24 hours using MEM-alpha supplemented with 20% human serum. Thereafter, the medium was changed to a low calcium serum-free medium (MCDB
153), and the culture was continued for about one week. The proliferating epidermal cells are detached and passaged by trypsin treatment,
Proliferation culture was performed using a low-calcium serum-free medium (MCDB153) and used for preparation of transplants (epidermal cells).

The dermis layer after the separation of the epidermis layer was washed with physiological saline, cut into small pieces, and MEMal supplemented with 20% human serum.
The cells were cultured in a pha medium for about one week, and the proliferating fibroblasts were subcultured three times (dermal fibroblasts). These cells were optionally supplemented with 20% human serum, 10% DMSO
Suspended in MEM Alpha, frozen in a program, stored in liquid nitrogen, and quickly thawed in warm water at 37 ° C before use.
After washing with each medium, the cells were cultured for a short period of time, and then used for preparing grafts.

Ethanol was added to fresh frozen plasma (manufactured by Nissekisha), left at −4 ° C. overnight, and centrifuged. The precipitate was dissolved in physiological saline (fibrinogen amount 400 mg).
/ Dl of crude fibrinogen solution). The cultured dermal fibroblasts were suspended in a very small amount of physiological saline, mixed with a crude fibrinogen solution, stirred sufficiently, and then placed in an incubator. Human thrombin (30 U / ml), calcium chloride (2 mM), PIPC ( 500 μg / ml) and urinastatin (5,000 U / ml), and left at 37 ° C. for several hours to obtain a fibrin sheet for transplantation having a thickness of about 1 mm in which dermal fibroblasts are dispersed and embedded. Was formed. The above-mentioned autologous epidermal cells were suspended in MCDB153 medium at a density of 2 × 10 ⁵ / cm ² and layered on the plate.
After culturing for 2 to 18 hours, an explant was prepared.

2. Transplantation Experiment The engraftment of the graft of the present invention was examined by the following method. First, immunodeficient mice (balb-c nu / s
The back skin of cid) was excised in full thickness with a size of 1.5 cm in diameter, and the above graft was implanted on the excised fascia and covered with a non-adhesive bandage. Subsequently, the implanted portion was visually observed and histologically examined over time.

The histological examination was performed by biopsy of the skin of the transplanted part over time, HE (hematoxylin-eosin) staining, HLA
Histological examination by immunostaining such as class I and electron microscopy was performed to determine whether or not the graft had survived. It was determined that the graft had survived macroscopically or that HLA class I-positive cells were observed. Performed as reference. The HLA class I positive cells were confirmed by immunostaining and gene analysis.

3. Results As a control test, only epidermal cells were allowed to adhere to the surface of the fibrin sheet, and a graft containing no fibroblasts and a composite graft prepared using collagen gel as a substrate were prepared, and a transplant test was performed. The results of the implant of the present invention are shown in Table 1 together with the results of these control tests.

[0040]

[Table 1] According to Table 1, when a graft to which epidermal cells and dermal fibroblasts were attached was used, the engraftment rate after transplantation was about 80%, and engraftment for 3 weeks or more was not observed with epidermal cells alone. Was. This means that when only epidermal cells were transplanted, normal human skin structure was not formed from the beginning, the basal structure was still immature, and epidermal atrophy was observed after that. Probably because it was difficult. Therefore, it can be seen that the graft of the present invention has a clearly superior engraftability as compared with the case where a graft consisting of only epidermal cells prepared according to JP-A-04-45785 is transplanted. In addition, the graft survival rate of the graft of the present invention was superior to that of the composite graft using collagen as a substrate. This is because, as detailed in the histological image below, fibrin implanted with cells has a higher biocompatibility and is more rapidly absorbed and disappears, and therefore is less likely to cause inflammation and infection than collagen. Is considered as one of the reasons. It can be easily inferred that this difference will be even more pronounced when transplanted into humans with a normal immune response.

FIGS. 1 and 2 show the macroscopic findings and the histological images of the reproduced tissues. FIG. 1 (a) is a reproduced HE (hematoxylin-eosin) stained image of the skin (3 weeks after transplantation), in which epidermal cells form a multilayer structure on the body surface, and a dermis-like structure Of connective tissue is formed. The basal layer, the spinous layer, the granular layer, and the stratum corneum are respectively observed in the epidermis, and although the epidermis is thickened, the tissue structure is similar to that of normal skin. An unstructured structure that is considered to be residual fibrin is observed under the epidermis. (B) shows HLA class I staining of the same specimen, but both epidermal and dermal cells are positive, indicating that the skin is composed of human cells.

FIG. 2 is (a) HE (hematoxylin-eosin) stained image of the reproduced skin (6 weeks after transplantation). The epidermis is thinner than that at 3 weeks after transplantation, and a tissue image close to normal skin is obtained. Present. Fibrin was not observed under the epidermis, and it is considered that it was absorbed and disappeared. In the type IV collagen immunostaining (b), the basal part (particularly, the epidermis-dermis junction)
In the subcutaneous connective tissue, local staining was observed in conformity with the luminal structure considered to be the endothelial basement membrane of the new blood vessel.

From the above, this graft can reproduce skin having a structure similar to normal human skin on mouse fascia tissue completely lacking skin elements, and fibrin used as a substrate can It can be seen that it is absorbed in about 6 weeks.

Embodiment 2 1. Clinical results In a clinical case of burn, this graft was transplanted to an old wound with full thickness skin defect.

A 78-year-old male. The bonfire ignited, causing a full thickness burn of the left lower limb (about 10% of body surface area). About 3 weeks after the injury, he was transferred to the hospital because of a wound infection. The wound skin was excised in full thickness up to the fascia, and thereafter the wound was treated with an antibacterial agent, an antibiotic or the like.

Approximately 7 weeks after the injury, debriefman was performed on the old granulation on the outside of the left lower limb under general anesthesia, and fibrin glue (Beriplast (trade name, manufactured by Boehringer)) was applied to the wound surface. Ten grafts (one piece of 75 cm ² ) produced by the following method were transplanted. It was covered with pentosillin-added vaseline ointment gauze and subjected to compression bandage. Transplant 5
Wounds were visually observed day after day, and a biopsy of the transplanted tissue was performed for histological examination.

2. Preparation of Grafts Separate 3 × 3 cm skin was aseptically collected from the patient's thigh and washed with sterile physiological saline. This is dispase
After treatment with II (2,000 U / ml) at 37 ° C for 20 to 40 minutes, the epidermal layer was separated and washed with physiological saline. 0.25%
The cells were treated with trypsin at 37 ° C. for 5 minutes and stirred in a serum-containing medium to disperse the epidermal cells. 5x dispersed epidermal cells
The cells were seeded in an incubator at a density of about 10 ⁴ / cm ² and cultured for 24 hours using MEM-alpha supplemented with 20% human serum. Thereafter, the medium was changed to a low calcium serum-free medium (MCDB15).
The culture was continued for about one week. The proliferating epidermal cells were detached and subcultured by trypsin treatment, expanded and cultured using a low-calcium serum-free medium (MCDB153), and used for preparing transplants (autologous epidermal cells).

The dermis layer after the separation of the epidermis layer was washed with physiological saline, cut into small pieces, and then MEMal supplemented with 20% human serum.
The cells were cultured in a pha medium for about one week, and the proliferating fibroblasts were subcultured three times (dermal fibroblasts). These cells were optionally supplemented with 20% human serum, 10% DMSO
Suspended in MEM Alpha, frozen in a program, stored in liquid nitrogen, and quickly thawed in warm water at 37 ° C before use.
After washing with each medium, the cells were cultured for a short period of time, and then used for preparing an explant.

After collecting ACD blood, ethanol was added to the centrifuged autologous plasma, the mixture was allowed to stand at −4 ° C. overnight, centrifuged, and the precipitate was dissolved in physiological saline (fibrinogen amount 40).
0 mg / dl crude fibrinogen solution). The cultured dermal fibroblasts were suspended in a very small amount of physiological saline, mixed with a crude fibrinogen solution, stirred sufficiently, and then placed in an incubator. Human thrombin (30 U / ml), calcium chloride (2 mM), PIPC ( 500 μg / ml) and aprotinin (250 U / ml) were added and left at 37 ° C. for several hours to obtain a fibrin sheet for transplantation having a thickness of about 0.5 mm, in which dermal fibroblasts were dispersed and embedded. Formed. The above autologous epidermal cells suspended in the MCDB153 medium were overlaid at a density of 5 × 10 ⁴ / cm ² , and 18
Cultured for hours. Thereafter, the fibrin sheet was peeled off from the incubator, and the graft was further suspended in a state where the graft was suspended in the medium.
After culturing for a time, it was used for transplantation.

3. Results At about 7 days after the transplantation, a structure that was visually confirmed as skin was formed at the transplant site. By the second week, about 90% of the wound area was covered with the reproduced skin, and the graft survival rate was considered to be about 90%. The wound was completely closed and healed 4 weeks after transplantation due to epithelialization from the graft.

FIG. 3 and FIG. 4 show macroscopic findings and histological images of the skin reproduced in the wound.

FIG. 3 shows macroscopic findings before transplantation (a) and 11 days after transplantation (b). About 90% of the wound area is covered by the skin reproduced after transplantation.

FIG. 4 is a histological image (HE-stained image) on day 9 after the transplantation, and a tissue similar to human skin with epidermal hyperplasia is observed. Neutrophil-based cell infiltration is observed under and inside the epidermis, but the tissue structure is not destroyed.

From the above, it can be seen that the present graft can reproduce a skin tissue having a structure similar to that of normal human skin when transplanted onto granulation tissue formed in a wound with a full thickness skin defect. Recognize. In addition, the grafts survived at a high rate even on the poorly transplanted beds of old granulation tissue formed in infected wounds of the elderly, and this graft was compared with conventional grafts consisting of cultured skin cells. As a result, it can be seen that the composition has extremely excellent adhesion.

[0055]

According to the present invention, skin and other squamous epithelial tissues can be reproduced at an excellent survival rate with respect to wounds on the body surface such as wounds in which the entire skin layer has been lost or squamous epithelial tissue in vivo. Thus, an implant is obtained that can permanently close the wound.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a drawing substitute photograph showing a tissue image of a skin tissue reproduced at a transplant site three weeks after transplantation. (A) shows a HE (hematoxylin-eosin) stained image, and (b) HLA class I antigen immunity. The stained image is shown.

FIG. 2 is a drawing substitute photograph showing a tissue image of a skin tissue reproduced at a transplant site 6 weeks after transplantation. (A) is a HE (hematoxylin-eosin) stained image, and (b) is a type IV collagen immunity image. The stained image is shown.

FIG. 3 is a drawing substitute photograph showing macroscopic findings of skin tissue reproduced at a transplant site before (a) and 11 days after transplant (b).

FIG. 4 is a drawing substitute photograph showing a tissue image of a skin tissue reproduced at a transplant site 9 days after transplantation, and is a HE (hematoxylin-eosin) stained image.

Claims (5)

[Claims] 1. A graft in which fibroblasts derived from epithelial tissue are embedded in a sheet of aggregated human plasma protein or aggregated human fibrin, and squamous cells derived from the epithelial tissue are attached to the surface of the sheet . 2. An implant in which fibroblasts derived from human dermis tissue are embedded in a sheet of aggregated human plasma protein or aggregated human fibrin, and epidermal cells are attached to the surface of the sheet. 3. The implant according to claim 1, further comprising a proteolytic enzyme inhibitor in the sheet of the aggregated human plasma protein or the aggregated human fibrin. 4. isolating squamous epithelial cells and fibroblasts from the epithelial tissue, and preparing squamous epithelial cells, epithelial tissues from which the squamous epithelial cells have been isolated, and fibroblasts derived from the same tissue, respectively. Proliferating and culturing the obtained squamous epithelial cells and the fibroblasts, embedding the proliferated and cultured fibroblasts in aggregated human plasma protein or aggregated human fibrin, Adhering squamous epithelial cells to the surface of aggregated human plasma protein or aggregated human fibrin having the cells embedded therein. 5. A step of isolating epidermal cells and fibroblasts from skin tissue and preparing epidermal cells and dermal-derived fibroblasts, respectively, and separating the obtained epidermal cells and fibroblasts respectively. Propagation and culturing, and embedment of the cultured and cultured fibroblasts in aggregated human plasma protein or aggregated human fibrin, and on the surface of aggregated human plasma protein or aggregated human fibrin in which the obtained fibroblasts are embedded. Adhering epidermal cells.