Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Animal or plant cell
Reexamination Certificate
1999-02-01
2003-07-01
Naff, David M. (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Animal or plant cell
C424S520000, C424S572000, C435S325000, C435S366000, C435S378000, C435S379000, C435S380000, C435S395000, C435S404000, C435S405000
Reexamination Certificate
active
06585969
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to new cultures of keratinocytes.
The invention also relates to a process for preparing the same.
The invention also relates to the use of new cultures of keratinocytes as wound healing substances.
The invention also relates to pharmaceutical compositions containing as active substances, said new cultures of keratinocytes.
The invention also relates to cosmetic compositions, containing as active substances, new cultures of keratinocytes.
DESCRIPTION OF THE PRIOR ART
Skin is presumably the organ most subject to injury. Skin repair is a complex process that can be divided in 4 phases usually described as inflammation, granulation tissue formation, epithelialization and remodeling of the connective tissue matrix. Each of these phases is complex in itself, and it is clear that for good wound healing, the processes must occur successively and in coordination. Good wound healing can be defined as restoration of the skin, including the dermal and epidermal part, in such a way that the resulting scar tissue maximally resembles the unwounded skin structurally, histologically, functionally, and esthetically obviously, such scar tissue is different from a hypertrophic scar or keloid.
For purposes of clarity a simplified description of the composition of human skin is given below. The upper part is composed of the epidermis, which contains mostly keratinocyte or epithelial cells, some melanocytes and Langerhans cells, and several Merkel cells. Five different layers are found in the epidermis, reflecting the state of keratinization. The proliferating keratinocytes at the base of the epidermis, i.e., in the stratum basal, are attached to the dermis via the basement membrane. The dermis is composed of connective tissue, including fibroblasts and other connective tissue cells, and connective tissue matrix substances. Blood vessels, nerves, sensory organs, sweat glands, sebaceous glands, and hair follicles are present in the dermis.
Clinical and animal experiments have demonstrated that application of in vitro cultured (human) keratinocytes, for instance as sheets, induces wound healing in chronic wounds such as ulcers and in burns, which may be treated concomitantly with meshed split skin autografts. Moreover, there are indications that the application of cultured keratinocyte grafts suppresses hypertrophic scar formation and keloid formation. Initially, autografts were used, which were prepared by growing keratinocytes isolated from the patients own skin. Confluent (differentiated) keratinocyte cultures are then detached from the culture dish and applied as a sheet, with basal cells facing downwards on the wound. About 3 weeks are needed before a reasonable amount of keratinocytes can be cultured for application as an autograft. Even then, the amount may not be sufficient to cover the entire wound surface. However, this time period may be critical for the patient, especially in the case of extensive third degree burns.
Therefore, experiments were undertaken to apply keratinocyte allografts. Keratinocytes are isolated from the skin of one person, cultured to obtain confluent keratinocyte sheets, and then applied on the wound of a patient. No differences in wound healing activity were observed between auto- and allografts. A main disadvantage of cultured allografts is the risk of transferring pathogens such as human immunodeficiency virus, hepatitis B virus, and cytomegalovirus, from the skin donor to the acceptor patient.
These techniques for wound treatment make use of fresh keratinocyte grafts, containing proliferating keratinocytes. The keratinocyte sheets are detached from the culture vessel by treatment with Dispase and immediately applied onto the wound. The availability of these sheets in terms of time and quantity is a major drawback of fresh keratinocyte grafts. It is assumed that proliferating keratinocyte cultures contain higher wound healing activity than more differentiated cultures. Moreover, only a multilayer culture can be detached as a sheet from the culture vessel, and can be spread over the wound. Therefore, cultured keratinocytes have to be taken at an optimal growth phase. However, it is impossible to predict when a patient will enter the hospital and how many keratinocyte grafts will then be needed. Another drawback of using fresh (autologous) keratinocyte sheets is that a certain period of time is still required before a sufficient amount of keratinocyte sheets can be grown and then applied.
Cryopreserved allografts partially circumvent these inconveniences. Cryopreserved keratinocyte grafts can be prepared as follows: confluent (differentiated) keratinocyte cultures are rinsed with phosphate buffered saline (PBS), cultured for 1 day without additives (epidermal growth factor, insulin, cholera toxin, triiodothyronin, transferrin, hydrocortisone), detached from the dish by treatment with Dispase and adhered to a transfer substrate (e.g., Interfaces Wuhrlin-Soplamed). The sheets are soaked in culture medium supplemented with 10% DMSO as cryoprotectant, and stored frozen in liquid nitrogen or for a shorter period of time at −60° C. Before application, the specimen is thawed and rinsed with PBS in order to remove DMSO and bovine serum proteins. The sheets are applied with basal cells facing downwards as fresh sheets. After thawing, about 60% of the cells are viable as judged from vital staining.
Initially, it was assumed that the keratinocytes from cultured allografts remain and grow on the wounds, an event which is clinically known as “take”. However, several reports recently demonstrated that wound closure is due to growth.of host epithelium.
Thus, healing is induced without permanent take of keratinocyte grafts, suggesting that the wound healing activity may be due to a (chemical) substance(s) associated with the cultured keratinocytes, which stimulates keratinocyte outgrowth. Consequently, it would not be necessary to apply sheets of viable keratinocytes. Therefore, the cultured keratinocyte sheets were lyophilized and the wound healing activity was compared with fresh and cryopreserved sheets.
Hence, the beneficial effect of cultured keratinocyte sheets in wound healing can probably be attributed to (a) substance(s) present in the cultured keratinocyte or induced by (a) molecule(s) present in the cultured keratinocytes. To date the(se) factor(s) and their concentration in the cultured keratinocytes are unknown. It cannot be excluded that certain known cytokines and growth factors are present. Many known growth factors, e.g., fibroblast growth factor (FGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor alpha (TGF-&agr;), transforming growth factor beta (TGF-&bgr;), influence keratinocytes and skin fibroblasts. For instance, TGF-&bgr;, EGF, and FGF induce keratinocyte proliferation. TGF-&bgr; and PDGF stimulate the synthesis of collagen and other connective tissue components. Vascularization can be influenced by basic-FGF and TGF-&agr;. Because of these properties, such growth factors may play a role in wound healing.
Clinical results showed that fresh and cryopreserved cultured keratinocyte sheets stimulate re-epithelialization, resulting in quicker healing compared to classic wound treatment or application of meshed split skin autografts only. According to the results of our animal model for wound healing, lyophilized cultured keratinocytes gave better results than keratinocyte sheets.
The term “lyophilized keratinocytes” denotes the product obtained from human or animal keratinocytes, which have been grown in vitro to the subconfluent, confluent, or differentiated state, and then a lyophilisate is prepared of these cells in such a way that no or minimal degradation of cell-associated substances occurs. Such a lyophilisate contains wound healing activity. Cell extracts from these cultured keratinocytes also contain wound healing activity. A cell extract may be prepared, for instance, after lysis and/or disruption of the cultured keratinocytes and subsequent fract
Merchant & Gould P.C.
N. V. Innogenetics S.A.
Naff David M.
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