Keratinocyte cultures and uses thereof

Details Keratinocyte cultures and uses thereof Keratinocyte cultures and uses thereof

2000-04-10

2004-05-04

Witz, Jean C. (Department: 1651)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Primate cell, per se

C424S093700

Reexamination Certificate

active

06730513

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to the field of cell culture of human keratinocyte precursor and dermal fibroblast cells. The invention also relates to the use of cultured keratinocyte precursor cells in the repair of skin defects by skin grafting procedures.
BACKGROUND OF THE INVENTION
The healing of skin defects progresses through three general phases: (i) inflammation, (ii) wound cell migration and mitosis, and (iii) extracellular matrix production and remodeling. The ordered sequence of these events is thought to be orchestrated by interactions among cells, growth factors, and extracellular matrix proteins. A crucial step of skin wound healing is epidermal regeneration (i.e., re-epithelialization). Besides interfollicular epidermal keratinocytes from the wound edges, the outer root sheath (ORS) cells from residual hair follicles also contribute to this process (see e.g., Eisen et al., 15 J. Invest. Dermatol. 145-155 (1955)). The ORS of hair follicles is comprised largely of undifferentiated keratinocytes that encompass the cylindrical structures of the hardened inner root sheath and the hair shaft (see e.g., Montagna & Parakkal, In:
The Structure and Function of Skin
172-258 (Academic Press New York, N.Y., 1974)). Recent literature has also indicated that ORS cells are at a lower level of commitment to differentiation than the basal interfollicular keratinocytes (see e.g., Coulombe et al., 109 J. Cell Biol. 2295-2312 (1989); Limat et al., 194 Exp. Cell Res. 218-227 (1991); Limat et al., 275 Cell Tissue Res. 169-176 (1994)), and label-retaining cells have been detected in the animal as well as the human ORS region near the bulge area which possibly represent stem cells for skin epithelial tissues (see e.g., Cotsarelis et al., 61 Cell 1329-1337 (1990); Kobayashi et al., 90 Proc. Nat. Acad. Sci. USA 7391-7395 (1993); Yang et al., 105 J. Invest. Dermatol. 14-21 (1993); Rochat et al. 76 Cell 1073-1076 (1994); Moll, 105 J. Invest. Dermatol. 14-21 (1995)). Additionally, human ORS cells which are isolated from plucked anagen scalp hair follicles can be expanded extensively in vitro (see e.g., Weterings et al., 104 Brit. J. Dermatol. 1-5 (1981); Limat & Noser, 87 J. Invest. Dermatol. 485-488 (1986); Imcke et al., 17 J. Am. Acad. Dermatol. 779-786 (1987); Limat et al., 92 J. Invest. Dermatol. 758-762 (1989)). Under conventional submerged culture conditions, ORS cells resemble interfollicular epidermal keratinocytes by both morphologic and biochemical (e.g., keratin profiles) criteria (see e.g., Stark et al., 35 Differentiation 236-248 (1987); Limat et al., 92 J. Invest. Dermatol. 758-762 (1989); Limat et al., 642 Ann. N.Y. Acad. Sci. 125-147 (1991)). In organotypic co-cultures with human dermal fibroblasts (i.e., under conditions mimicking the epidermal environment), ORS cells with respect to histological, immunohistological, ultrastructural and biochemical criteria develop a stratified epithelium reminiscent of regenerating epidermis (see e.g., Lenoir et al., 130 Dev. Biol. 610-620 (1988); Limat et al., 194 Exp. Cell Res. 218-227 (1991); Limat et al., 642 Ann. N.Y. Acad. Sci. 125-147 (1991)). If such organotypic cultures are grafted onto nude mice, ORS cells form a regular neo-epidermis that is under homeostatic control (see e.g., Limat et al., 59 Transplantation 1032-1038 (1995)). Thus, human ORS cells are of considerable interest for clinical application.
In the previous decade, interest has focused on the use of cultured epithelial cells for wound coverage. First, sheets of cultured autologous interfollicular keratinocytes were grafted successfully on acute wounds, mainly in the treatment of larger third degree burns (see e.g., O'Connor et a., 1 Lancet 75-78 (1981); Compton et al., 60 Lab. Invest. 600-612 (1989)) but also of epidermolysis bullosa (see e.g., Carter et al., 17 J. Am. Acad. Dermatol. 246-250 (1987)), pyoderma gangrenosum (see e.g., Dean et al., 26 Ann. Plast. Surg. 194-195 (1991); Limova & Mauro, 20 J. Dermatol. Surg. Oncol. 833-836 (1994)), and wounds after excision of giant congenital nevi (see e.g., Gallico et al., 84 J. Plast. Reconstr. Surg. 1-9 (1989)) or separation of conjoined twins (see e.g., Higgins et al., 87 J. Royal Soc. Med. 108-109 (1994)).
In contrast to the treatment of such acute wounds, the grafting of chronic wounds (e.g., leg ulcers) with cultured keratinocytes has been much less successful. Allografts do not result in a permanent “take” (see e.g., Fabre, 29 Immunol. Lett. 161-166 (1991)) and thus may be classified as a “quite effective but expensive biological dressing” (see Phillips et al., 21 J. Am. Acad. Dermatol. 191-199 (1989). A reproducible, major definite “take” of autologous keratinocyte grafted by various modalities including: sheets of submerged keratinocyte cultures consisting of only a few, noncornified cell layers (Hetton et al., 14 J. Am. Acad. Dermatol. 399-405 (1986); Leigh & Purkis, 11 Clin. Exp. Dermatol. 650-652 (1986); Leigh et al, 117 Brit. J. Dermatol. 591-597 (1987); Harris et al., 18 Clin. Exp. Dermatol. 417-420 (1993)), trypsinized single cells attached to collagen-coated dressings (Brysk et al., 25 J. Am. Acad. Dermatol. 238-244 (1991)), skin equivalents (Mol et al., 24 J. Am. Acad. Dermatol. 77-82 (1991)) has yet to be convincingly documented within the scientific literature. The same lack of quantitative findings also holds true for various reports on the grafting of freshly isolated, autologous interfollicular keratinocytes (Hunyadi et al., 14 J. Dermatol. Surg. Oncol. 75-78 (1988)) or ORS cells (Moll et al., 46 Hautarzt 548-552 (1995)) fixed to the wound bed by the use of a fibrin glue. However, it should be noted that the disadvantages of the bovine serum used during cultivation of the keratinocytes may contribute to reduced “take” rate, due to the fact that it resists in keratinocytes (see e.g., Johnson et al, 11 J. Burn Care Rehab. 504-509 (1990)).
SUMMARY OF THE INVENTION
Prior to the disclosure of the present invention herein, the standard methodology for the generation of a primary culture of ORS keratinocytes consisted of the plucking of an anagen (i.e., growing hair shaft) hair followed by a careful microscopic dissection to remove the hair bulbs and the infundibular hair shaft. The resulting outer root sheath was then placed on the culture insert for initiation of the primary keratinocyte culture. However, numerous subsequent studies (approximately 200), wherein the anagen hair was placed directly on the culture insert without performing the initial micro-dissection to remove the hair bulbs and the infundibular hair shaft, have demonstrated that such tedious and time-consuming dissection of the plucked anagen hair was not required. This has served to markedly simplify the handling process, reduce the risk for contamination, and resulted in more efficient initiation of keratinocyte cell plating.
Accordingly, it is an object of the present invention to provide improved and simplified methods for the generation of keratinocytes or keratinocyte precursors from outer root sheath cells (ORS cells) in fully defined culture conditions for the treatment of various types of skin defects (e.g., chronic wounds such as leg ulcers, diabetic ulcers, pressure sores, and the like) in both humans and animals. In addition to their use in the treatment of wounds, keratinocytes may also be used in plastic and cosmetic surgery, or whenever there is a demand for such skin support (e.g., post operative following the removal of tattoos, naevi, skin cancer, papillomas, after amputation, in sex transformation or re-virgination, rejuvenation of actinically damaged skin after skin resurfacing, tympanoplasty, epithelialization of external ear canal, and the like).
These aforementioned objectives are accomplished by explantation and culture of plucked, anagen or growing hairs in toto upon microporous membranes carrying human fibroblast feeder cells at their under-surface. In such primary cultures, large numbers of ORS cells can be easily and repeatedly obtained, irrespective of the donor's chronological age.

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