WO1990001266A1

WO1990001266A1 - Transplantig cultured epithelial cells with a desired phenotypic trait

Info

Publication number: WO1990001266A1
Application number: PCT/US1989/003372
Authority: WO
Inventors: Carolyn C. Compton; Howard Green
Original assignee: President And Fellows Of Harvard College
Priority date: 1988-08-04
Filing date: 1989-08-04
Publication date: 1990-02-22
Also published as: JPH04501054A; EP0430996A1; AU4473793A; EP0430996A4; AU4061989A

Abstract

Tissue produced by culturing isolated epithelial cells (preferably squamous cells) will retain the ability to express the phenotypic characteristics of the isolated cells when the tissue is transplanted into a new region of body surface. Thus defects in the epithelium (e.g., burns, ulcers and congenital abnormalities) can be treated by obtaining a sample of living epidermal cells from a region of the patient's epithelium (e.g., sole of the foot, palm of the hand or scalp) having a site-specific phenotypic characteristic (e.g., a thick stratum corneum or a stratum lucidum positioned between the stratum granulosum and the stratum corneum) which is desired for the graft at the defective region. These epidermal cells are cultured, according to methods well-known to those skilled in the art, to produce transplantable living cells, e.g. an aggregation or sheet of living tissue. This tissue is then transplanted to the site of the defect where it expresses the phenotypic characteristic in a region of the patient's epithelium that may or may not ordinarily manifest this characteristic.

Description

TRANSPLANTING CULTURED EPITHELIAL CELLS WITH A DESIRED PHENOTYPIC TRAIT

Background of the Invention This invention relates to methods of treating defects in the epithelium of a patient, e.g. burns, ulcers, bedsores, or congenital abnormalities.

Previous methods of treating epithelial defects involved grafting a patch of skin from a healthy donor site directly to the defect. However, for patients with extensive defects, e.g., massive burn wounds, there is a limited availability of donor sites for skin grafts to close the wounds, and it is undesirable to exacerbate the loss of skin by removing healthy skin to be grafted. Recent advances in the in. vitro cultivation of keratinocytes—the major cellular constituent of the epidermis—have made it possible in a matter of weeks to generate enough epidermis from a small donor biopsy to cover substantial body surface. In particular, Green et al., U.S. Patent No. 4,016,036, disclose techniques to grow keratinocytes in cultures in the presence of fibroblast cells which are treated to prevent their multiplication. Green and his co-workers demonstrated that fibroblast cell products support the growth of keratinocytes. However, fibroblast cell density was controlled to allow epidermal cell colony formation and growth.

In a more recent patent, Green et al . , U.S. Patent No. 4,304,866, disclose a method for producing transplantable sheets of living keratinous tissue (e.g., epidermal cell tissue). According to this method, keratinocytes are cultured to form a sheet on a vessel surface. The keratinous tissue is treated with a neutral protease to detach the tissue from the surface of the vessel. The teachings of both patents are incorporated herein by reference. Morgan et al., WO87/00201, generally disclose genetic engineering of cultured epidermal cells to express foreign proteins.^' In one embodiment, they disclose formation of a skin graft from a biopsy of keratinocytes (e.g., epidermal cells) into which foreign DNA is introduced and expressed to alter the phenotype of the cultured cells. For example, they introduce the gene for a protein (keratin-9) that is characteristic of plantar cells into keratinocytes with the following goal.

"Once ... [the] gene [for the keratin protein] is cloned, it can be introduced into a skin transplant that could be placed in an area where a tougher skin is desirable. For example, this might be useful in the case of an immobilized patient, for whom bed sores might be a severe problem. "

Morgan et al. do not indicate the region from which the biopsy of keratinocytes should be obtained.

Summary of the Invention We have discovered that tissue produced by culturing isolated epithelial cells (keratinocytes) retains the phenotypic characteristics of the isolated cells, and will retain those characteristics when the tissue is transplanted to a different region of the body surface to generate epithelium.

In general, one aspect of this invention features culturing tissue to be used for treating defects (e.g., burns, ulcers or congenital abnormalities) in which there is loss or absence of epithelium of a patient. A region of epithelium containing epithelial cells having a desired site-specific phenotypic characteristic is identified, and living epithelial cells from that region are obtained and cultured according to known techniques to produce transplantable living cells, which have the ability (upon transplantation to the defect site) to express that phenotypic characteristic (e.g., a thick stratum corneum and a stratum lucidum positioned between the stratum granuloεum and the stratum corneum) which is desired for the graft at the defective region.

In preferred embodiments, the culture results in a transplantable aggregation ^"of cells, e.g. at least one sheet of living tissue, which is transplanted to the defect site. The epithelial cells to be cultured can be (but need not be) obtained from a region of the epithelium other than the region of the defect site; and they can be obtained from a region which has the same or different phenotypic characteristics from those of the defect site.

Cultures generated from epidermal cells from the of sole of the foot or the palm of the hand yield tissue capable of particularly rapid generation of rete ridges, which aid interaction with tissue beneath the skin culture, i.e., the dermal-epidermal junction, to root the culture more solidly. For example, sole-derived cultures have been observed to form rete ridges as rapidly as six weeks after transplantation, as compared to several months for cultures derived from other sites. For that reason alone, such cultures are desirable. Moreover, such cultures are also desirable because, once transplanted, the tissue retains the sole-skin or palm-skin phenotypic characteristics. including a thick stratum corneum and a stratum lucidium between the stratum granulosu and the stratum corneum. These characteristics are particularly desirable for defects at sites which must tolerate friction and pressure. Thus, preferred embodiments of the first aspect of the invention feature obtaining human epithelial cells from the sole of the foot or the palm of the hand and culturing them as described above.

A second aspect of the invention features a method of culturing tissue by obtaining living human epithelial cells from the sole of the foot or the palm of the hand, and then culturing those epithelial cells to produce transplantable cells.

A third aspect of the invention features obtaining living human epithelial cells from the sole of the foot, culturing those cells to produce transplantable living cells, and transplanting the aggregation to the epithelial defect, whereby the transplant rapidly forms rete ridges. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Description of the Preferred Embodiments The methods of this invention are particularly well-suited for the treatment of defects in a patient's epithelium such as burns, ulcers (e.g. bedsores), and congenital abnormalities, whenever it is desirable to graft tissue having a known site-specific phenotypic characteristic. The invention can be practiced in various specific embodiments. Without limiting the invention, two specific situations in which the invention can be used are described below. Transplanting Cultured Epidermal Cells From The Sole Of The Foot

It is common for bedridden patients to develop ulcers (e.g., bedsores) on the backs of their legs and their buttocks. In many instances, these patients are treated with skin grafts which are also prone to ulceration. This invention provides a-method of transplanting an epidermal tissue specimen having the predetermined phenotypic characteristic of a thick stratum corneum and is thus, a type of epidermal tissue which is particularly resistent to bedsores. Epidermal cells which are known to have a thick stratum corneum include those cells from the bottom of the foot (plantar region), and the palm of the hand (palmar region). Thus, epidermal cells from either the plantar or, almar regions can be cultured to produce sheets of transplantable tissue having a thick stratum corneum. Such tissue can be transplanted to an ulcer on the buttocks of the patient, where this new tissue will retain the phenotypic characteristic of a thick stratum corneum.

Scalp skin is also relatively tough. Regions of a patient's body which are susceptible to ulceration will be protected by a thick skin like that found on the bottoms of one's feet, the palms of one's hands, or one's scalp.

Specifically, skin is harvested, e.g. from the sole of the foot, by techniques known to those in the field, such as dissection of the skin with a scalpel blade or a punch biopsy. The grafts are prepared generally by the method of Green et ai . , Proc. Na ' 1. Acad. Sci. (USA) 7_6: 5665-5668 (1979) and Green et al. U.S. Patent 4,016,036. More specifically, the sole skin biopsies are placed aseptically-into fortified Eagles medium containing 10% calf serum, at room temperature. Single cells are obtained by mincing, stirring in trypsin at 37°C, and withdrawing supernatant containing single cells.

The cells are centrifuged, resuspended in medium containing 10% fetal calf serum and hydrocortisone (0.4μg/ml), mixed with lethally irradiated 3T3 cells and plated. The medium is changed 3-5 days later when most epidermal cells had attached and twice weekly thereafter. Confluent cultures can be detached with dispase and grafted to sites where sole skin characteristics are desired. The resulting grafts will retain those characteristics.

Cells from the sole of the foot produce relatively few primary colonies, but they produce very rapid growth in secondary colonies, yielding confluent colonies in less time.

Transplanting Keratinocytes of Wet Surface Epithelia

Where the site of the epithelial defect includes mucous tissue, e.g. a vaginal reconstruction or a site within the mouth as in a repair of a cleft palate, it may be desirable to obtain cells from a site that also has wet epithelium. In this way, the phenotypic traits adapted to existence in the wet environment can be preserved in the graft.

The technique for transplanting epithelial cells derived from wet surface epithelium (preferably squamous epithelium) is essentially identical to the technique described above for sole skin transplants. The inside of the mouth is a suitable source for cells to be cultured. Other Embodiments Other embodiments are within the following claims. For example, skin from the palm of the hand can be used instead of plantar skin from the sole of the foot. Scalp skin is also relatively tough. The defects treated include congenital abnormalities, e.g. cleft lip, reconstruction after surgery, e.g. vaginal reconstruction, treatment for burns, bed sores, ulcers etc. The treatment of a congenital abnormality, such as a cleft lip, may require reconstructive surgery and/or tissue transplants. This invention provides a method of transplanting an epidermal tissue specimen having the particular phenotypic characteristics which are best suited for facial surgery. Clinicians who are skilled in the art will know, or will be able to determine without undue experimentation, those epidermal cells having phenotypic characteristics best suited for the treatment of the particular abnormality. While the preferred culturing method is described above, other cell culture techniques can be used in the invention, such as the techniques described by Boyce and Ham in J. Tissue Culture Methods 9 (2) : 83-93 (1985) or Eiεinger et al . Proc . Acad. Sci. USA 7> (10) : 5340-5344 (1979). While the preferred technique involves formation of transplantable sheets of tissue, non-sheet-forming techniques can be used, in which the transplantable cultured cells are a suspension of single cells, or they are a matrix containing single cells. See generally Yannas and Burke in U.S. Pat. 4,458,678 regarding such a matrix.

Claims

Claims 1. A method for culturing tissue comprising obtaining living human epithelial cells from the sole of the foot or the palm of the hand, and culturing said epithelial cells to produce transplantable living cells.

2. A method for culturing tissue comprising obtaining living human epithelial cells from the sole of the foot, culturing said epithelial cells to produce transplantable living cells, and transplanting said cultured cells to a defect in the epithelium of a patient, whereby said transplant rapidly forms rete ridges.

3. A method for culturing tissue to be used in treating a defect in the epithelium of a patient, said method comprising: identifying a region of said epithelium containing epithelial cells having a desired site-specific phenotypic characteristic; obtaining living epithelial cells from said region; and culturing said living epithelial cells to produce transplantable living cells, said cultured cells having ability upon transplantation to express said phenotypic characteristic.

4. The method of claim 1, 2, or-3 wherein said culturing produces a transplantable aggregation of living cells.

5. The method of claim 1 or claim 3 further comprising transplanting said cultured living ceils to a defect in the epithelium of said patient.

6. The method of claim 4 wherein said transplantable aggregation of living cells comprises a transplantable sheet of living tissue.

7. The method of claim 1, 2, or 3 wherein said transplantable living cells comprise a suspension of single cells, or a matrix containing single cells.

8. The method of claim 5 wherein said living human epithelial cells are obtained from a first region of the patient's epithelium, and said living tissue is transplanted to a defect at a second region of said patient's epithelium.

9. The method of claim 5, wherein said defect is a burn, an ulcer, or a congenital abnormality.

10. The method of claim 2 wherein said defect is a burn, an ulcer or a congenital abnormality.

11. The method of claim 3, wherein said desired phenotypic characteristic is a thick stratum corneum.

12. The method of claim 3, wherein said desired phenotypic characteristic is the presence of a stratum lucidum positioned between the stratum granulosum and the stratum corneum.

13. The method of claim 3, wherein said living epithelial cells are obtained from the sole of the foot, the palm of the hand or the scalp.

14. The method of claim 3, wherein said living epithelial cells are obtained from wet epithelium, and said living tissue is transplanted to a defect in wet epithelium of said patient.

15. A method of treating a burn in the epithelium of a patient, comprising: obtaining living epithelial cells from the plantar or the palmar or the scalp region of said epithelium; culturing said living epithelial cells to produce transplantable living cells; and transplanting said tissue to the burn site of said patient, whereby said transplanted tissue generates thick stratum corneum.