Link: Pharm/Biotech Resources
Title: Cultured skin and method of manufacturing the
same
United States Patent: 6,916,655
Issued: July 12, 2005
Inventors: Yasumoto; Shigeru (Kanazawa-ken, JP); Takeuchi; Masakatsu (Osaka, JP)
Assignee: Nipro Corporation (Osaka, JP)
Appl. No.: 300906
Filed: November 21, 2002
Abstract
A cultured skin and a grafting cultured skin sheet are provided, each of which is a cultured reconstructive skin with a high take rate using cells collectable from cells originated from tissue included in an umbilical cord such as tissue included in an umbilical cord originated from a human fetus. The grafting cultured skin stratified sheet is prepared by placing an epithelium sheet on the top surface of a cultured dermis. The cultured dermis includes as components a cultured skin containing cells originated from a tissue included in an umbilical cord, such as umbilical cells, more concretely, umbilical fibroblast cells, being separated and cultured, preferably in a collagen nonwoven fabric. On the other hand, the epithelium sheet is prepared by culturing and stratifying the umbilical cord epithelium cells.
SUMMARY OF THE INVENTION
The present inventors have made dedicated efforts to provide a cultured
reconstructive skin with a high successful grafting rate using cells
collectable from tissues included in an umbilical cord originated from a
human fetus, finally completing the present invention.
That is, the present invention relates to:
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, the term "cells originated from tissue included in an umbilical cord" refers to, for example, umbilical cells, placenta cells, velamen cells, and so on. The umbilical cells are constitutive cells of an umbilical cord. The umbilical cord is a human fetal appendage and becomes unnecessary after birth. However, it can be provided as a successive source of supplying fetal cells that retain potential self-regenerative activities and capacity for differential plasticity. The placenta cells are those of an elliptically squamous organ comprising fetal chorion frondosum and maternal decidua basalis. The fetal side of the organ is covered with the amnion. The velamen cells are those of components of a membrane that enwraps an embryo or the like in a uterus, which are formed from decidua, chorion, and amnion in that order from the outer layer. The amnion is a semi-translucent thin film that covers the fetal side of the placenta and the inside of the velamen. An epithelium of the amnion indicates a shift to the surface of umbilical cord, i.e., an epithelium tissue on the umbilical cord and fetal skin portion. The amnion consists of five layers including a mono layer of an epithelium cell layer and a fibroblast layer, while the chorion consists of four layers including a fibroblast layer.Umbilical cells to be used in the present invention include umbilical epithelium cells and umbilical myofibroblasts separated from umbilical tissue.
In general, umbilical tissue (prenatal fetal tissue) is comprised of an umbilical epithelium (including an external layer), Wharton's jelly that involves umbilical myofibroblasts, two umbilical arteries, and one umbilical vein. In the body, the umbilical tissue is in a growth process, and also the umbilical epithelium is a part of epithelial tissue of a body surface. Therefore, it is a tissue that contains many regenerative epithelial stem cells without a progress in aging, as distinct from adult skin. This can be supported by a high telomerase activity and a long telomere length. The division lifetime (aging) of the cell can be delayed by such telomerase activity and allows the preparation of graft skin with a long term take.
Furthermore, the cellular aging delay effect can be easily exerted by the introduction of a cell-immortalizing gene. Also, it is known that such an effect of the above cell where a cell-immortalizing gene is introduced is extremely higher than that of adult skin cells (up to several hundred times). From this fact, it is clear that the potential division abilities of umbilical epithelium cells are extremely high.
However, the introduction of an immortalizing gene involves a modification of cells. Thus, there is no restraint on the preparation of cultured skin sheet using immortalizing umbilical epithelium with respect to a combination with an operation of removing the transgene from the cell. Further, implementation thereof does not restrict application of using an adeno-cre-Lox system known in the art.
Furthermore, the surface layer of the umbilical cord is connected to the surface skin of a fetus, so that there is a possibility that the cells included in the umbilical cord have the same or more excellent regenerative abilities, compared with those of adult skin cells.
Moreover, as those cells are originated from a fetus, there are further advantages in that the lifetime of cell division can be longer than others and a high potential growth ability can be attained.
The umbilical epithelium cells to be used in the present invention are those grown by separating columnar epithelium cells in about one to three layers of the epithelium layers that cover the umbilical outermost layer, followed by culture.
The method of separating the umbilical epithelium cells includes the following steps of:
(1) immersing a tissue included in an umbilical cord in a proteolytic enzyme to loosen the binding of the tissue included in the umbilical cord;
(2) peeling the tissue included in the umbilical cord by physical means; and
(3) separating the peeled tissue into cells by the proteolytic enzyme.
Preferably, for obtaining the umbilical epithelium cells of the present invention, a piece of epithelium tissue is separated by treating the tissue included in the umbilical cord such as an umbilical tissue with a proteolytic enzyme or the like and the resultant tissue is further broken into cells by treating with a solution of trypsin-EDTA or the like. The proteolytic enzyme may be dispase, trypsin, collagenase, or the like, which is an enzyme capable of degrading fibronectin and collagen type I or IV. In the step of physically peeling the umbilical tissue (a piece epithelium tissue), instruments such as a scalpel, a pair of tweezers, and a brush are used.
According to the present invention, the medium for the culture of umbilical epithelium cells in the present invention is not specifically limited. However, one of media including those used for epithelium cells other than umbilical epithelium cells or fibroblasts may be used. Such media include those for animal cells, such as MCDB153 (Sigma), keratinocyte-SFM (GIBCO), Defined keratinocyte-SFM (GIBCO), EpiLife-KG2 (Kurabo), HuMedia-KG2 (Kurabo), HuMedia-KB2 (Kurabo), DMEM (Sigma), RPMI1640 (GIBCO), Medium 106S (Kurabo), and so on. Among them, it is preferable to prepare and use a medium (MCDB153-1/4) as a mixture of the MCDB153 medium (including supplements) and the DMEM medium (including 0-10% FBS) at a volume ratio of 4:1.
The culture vessels to be used in the present invention include culture dishes, culture flasks, membranes, cover glasses, and so on. Typically, they are made of polystyrene, glass, polypropylene, TC-treated polycarbonate, blended cellulose ester, hydrophilic-treated PTFE (IWAKI, NUNC, CORNING, FALCON), or the like. In addition, preferably, each of their surfaces may be coated with an extracellular adhesive material. The extracellular adhesive material may include one selected from the group consisting of collagen, laminin, elastin, proteoglycan, tenascin, and fibronectin. The epithelium cells maintain contact with the connective tissue under them through a basal lamina. Therefore, it is preferable to use a culture vessel coated with collagen type I or IV, which is a component of the basal lamina.
The culture conditions to be used in the present invention are preferably of about 37° C. and 100% humidity in the presence of 5% carbon dioxide. However, the present invention is not limited to these conditions.
The umbilical myofibroblasts to be used in the present invention are those grown by separating myofibroblasts included in Wharton's jelly in the umbilical cord, followed by the culture thereof.
The umbilical fibroblasts may be prepared by a method in which fibroblasts in the tissue included in the umbilical cord are separated and cultured to allow the growth thereof.
The method of separating the umbilical fibroblasts may be one in which umbilical fibroblasts are separated from the tissue included in the umbilical cord, such as an umbilical tissue by means of an explant culture method.
The explant culture method is one for culturing a small piece of tissue taken from the living body, allowing the collection of cells liberated and grown from the tissue.
The umbilical myofibroblasts separated from the umbilical tissue can be cultured in the medium in a culture vessel. More concretely, the umbilical myofibroblasts may be preferably separated and cultured using an explant culture method after separating the umbilical epithelium cells and removing blood vessels within the umbilical cord from the umbilical tissue. Concretely, for example, the umbilical tissue is cut out into small tissues of about 1 to 5 mm squares using a scalpel or the like, followed by leaving them at rest in a culture vessel containing the DMEM medium (including 10% of FBS) for 5 to 10 days. Then, the umbilical myofibroblasts being migrated and divided and grown from the periphery of the tissue are separated using a solution such as trypsin-EDTA or the like, followed by the culture of the thus-obtained cells in the DMEM medium (including 10% of FBS) for 5 to 30 days.
The medium to be used for umbilical myofibroblasts of the present invention is snot specifically limited. However, for example, DMEM (Sigma), RPMI1640 (GIBCO), and Medium106S (Krabo) can be exemplified. Among them, especially, the DMEM medium (including 10% FBS) is preferable.
The culture vessels to be used in the present invention include culture dishes, culture flasks, cover glasses, and so on. The culture conditions to be used in the present invention are preferably of about 37° C. and 100% humidity in the presence of 5% carbon dioxide. However, the present invention is not limited to these conditions.
The cultured skin to be prepared in the present invention is a two-layered cultured skin prepared using a skin of the living body as a model, where the skin includes an epidermal layer and dermal layer and umbilical cells are included as components of the epidermal and dermal layers.
Among the umbilical cells, preferably, umbilical epithelium cells may be in the epidermal layer, while umbilical myofibroblasts may be in the dermal layer. The epidermal layer can be prepared by stratifying 4 to 10 layers of the umbilical epithelium cells. On the other hand, the dermal layer can be prepared using the umbilical myofibroblasts as carriers, preferably together with an extracellular matrix such as collagen.
The epithelium sheet to be prepared in the present invention is a cultured epithelium prepared using the epithelium layer of the skin of the living body as a model. Preferably, it may be prepared by stratifying 4 to 10 layers of the umbilical epithelium cells.
The cultured dermis to be prepared in the present invention is one preferably prepared using the dermal layer of the skin of the living body as a model, while the umbilical myofibroblasts are used as carriers, preferably together with an extracellular matrix such as collagen. In a specific testing method, when the fibroblasts are only grafted in an in vitro environment, such fibroblasts are multiplied so that collagen can be produced. As a result, a dermis-like tissue is formed. Typically, in the in vitro environment, the dermal layer cannot be formed using the fibroblasts only.
As the extracellular matrices, collagen non-woven fabric, collagen gel, collagen sponge, collagen sheet, and so on can be exemplified.
Instead of these carriers, alternatively, bio-absorbable substrates (glycosaminoglycan, glycolic acid, lactic acid, chitin, polyglactin, chondroitin sulfate, and fibrinogen) and so on may be used.
The method of manufacturing cultured dermis using the carrier may be one in which fibroblasts are inoculated in the carrier or inoculated on the carrier to allow a successful grafting, followed by culturing for 1 to 4 weeks. In the case of gel carrier, the carrier in a liquid form is mixed with fibroblasts and is then gelated and cultured for 1 to 4 weeks to prepare cultured dermis that includes fibroblasts in the gel.
Preferably, the cultured dermis is of 0.5 to 3 mm in thickness and is provided in the shape of a smooth sheet.
A grafting cultured skin stratified sheet of the present invention is prepared by placing an epithelium sheet on the surface is of a cultured dermis which includes umbilical fibroblasts. That is, a grafting cultured skin stratified sheet of the present invention is prepared by a process that separated and cultured umbilical epithelium cells are further cultured and stratified on the surface of a cultured dermis including separated and cultured umbilical fibroblasts as components thereof.
Alternatively, a grafting cultured skin stratified sheet of the present invention is prepared by placing an epithelium sheet on the surface of a dermal layer consisting of collagen gel prepared from a collagen solution and umbilical fibroblasts. That is, a grafting cultured skin stratified sheet of the present invention is prepared by a process that separated and cultured umbilical epithelium cells are further cultured and stratified, on the surface of a dermal layer consisting of collagen gel prepared from a collagen solution and umbilical fibroblasts.
In the case that the separated and cultured umbilical epithelium cells are cultured and stratified on the surface of the cultured dermis or the dermal layer, separated umbilical epithelium cells or umbilical epithelium cells previously cultured once are inoculated and cultured on the surface of the cultured dermis or the dermal layer, followed by further stratifying. This kind of stratified structure can be attained by continuing the culture under conditions including the addition of an epithelium cell differentiation factor and air exposure of the epithelium layer.
Generally, stratifying includes the steps of inoculating umbilical cord epithelium (epithelial) cells on the dermal layer, culturing for about 4 to 10 days, preferably at 37° C. in the presence of 5% CO2, and exposure of the epithelium (epithelial) layer to air.
In the grafting cultured skin stratified sheet, the dermal layer has a thickness of 0.5 to 3 mm in general, the number of epithelium layers is 4 to 10 with a thickness of 0.1 to 1 mm in general.
The dermal layer and the epithelium layer may further include vascular endothelial cells, nerve cells, hematopoietic stem cells, blood vessel inducer, epithelium cell differentiation factors, epithelium cell growth factors, and fibroblast growth factors.
The cultured skin of the present invention has a high potential division ability and a comparatively low antigenicity, and is mainly constructed of cells of fetal origin umbilical tissue, which can be successively supplied, so that it can be applied in medical treatments on diabetic leg ulcers, severe burns, and so on.
Claim 1 of 1 Claim
1. A method of culturing umbilical cord epithelium cells, characterized by
culturing in a medium umbilical cord epithelium cells separated from cells
originated from tissue included in an umbilical cord, wherein the medium
is a mixture of MCDB153 medium and DMEM medium in a volume ratio of 4:1
supplemented with epidermal growth factor, insulin, transferrin,
hydrocortisone, ethanol amine, phosphoethanolamine and hypophysis extract.
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