Assessment of transformed properties in vitro and of tumorigenicity in vivo in primary keratinocytes cultured for epidermal sheet transplantation (original) (raw)
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Mayo Clinic Proceedings, 1986
Human keratinocytes can be cultured in vitro and used for autografting of the injured epidermis. In this article, w e describe our culture methods for propagating autologous keratinocytes for grafting, and w e present an overview of the questions raised by the use of such methods. Our culture method involves a two-phase technique. Phase 1 is performed in serum-free medium and yields large quantities of a homogeneous, highly proliferative, basal cell-like population. Phase 2 involves the induced formation of cohesive, stratified sheets of differentiated keratinocytes resembling normal epidermis that can be used for grafting. This two-phase technique w a s used for preparation of autografts for full-thickness wounds of a massively burned patient. Important questions about the functional potential of cultured keratinocytes and the long-term behavior of cultured epidermal autografts in vivo remain to be answered.
Experience gained during the long term cultivation of keratinocytes for treatment of burns patients
Cell and Tissue Banking, 2011
Both allogenic and autologous cultured skin cells have been used clinically on burn patients. In vitro cultivation of human keratinocytes has been routinely provided by the Central Tissue Bank in Bratislava since 1996, with an average annual production of around 7,000 cm 2. Keratinocytes have been cultivated using a version of the original by Rheinwald and Green (Cell 6:317-330, 1975) methodology which has been modified over time in our laboratory as we gained more experience with this serial passage system. We have observed that the growth of cultured keratinocytes depends on several important factors, including the timing of skin sample procurement, the method of skin sample procurement, the general condition of the patient, the quality and composition of the culture media and, to a lesser extent, the age of the patient. We aim to share our experience with other cell cultivation facilities.
An alternative approach for traditional clinical mesh grafting in burn wound treatment is the use of expanded autologous keratinocytes in suspension or sheets that are cultured over 2–4 weeks in a remote service facility. While a wound reepithelialization has been described, the functional and aesthetic outcome is under debate. Cell isolation from split-skin donor tissue aims to preserve the valuable stem cell progenitors from the basal epidermal layer and to provide patients with a rapid wound re-epithelialization and a satisfying outcome. While the presence of epidermal progenitors in the cell graft is thought to enable an improved epidermal surface post reepithelialization, we investigated a feasible clinical approach involving cultured versus noncultured epidermal cells comparing the α6int high /K15 high /FSC low /SSC low and α6int high /K5 high / FSC low /SSC low keratinocyte progenitor subpopulations before and after in vitro culture process. Our results show a significant increase of cell size during in vitro passaging and a decrease of progenitor markers linked to a gradual differentiation. A provision of the regenerative epidermal progenitors, isolated from the split-skin biopsy and applied directly onto the wound in an on-site setting of isolation and cell spray grafting in the operation room, could be of interest when choosing options for skin wound care with autologous cells.
Culture of Keratinocytes for Transplantation Without the Need of Feeder Layer Cells
Cell Transplantation, 2007
Patients with large burn wounds have a limited amount of healthy donor skin. An alternative for the autologous skin graft is transplantation with autologous keratinocytes. Conventionally, the keratinocytes are cultured with mouse feeder layer cells in medium containing fetal calf serum (FCS) to obtain sufficient numbers of cells. These xenobiotic materials can be a potential risk for the patient. The aim of the present study was to investigate if keratinocytes could be expanded in culture without the need of a feeder layer and FCS. Keratinocytes were cultured on tissue culture plastic with or without collagen type IV coating in medium containing Ultroser G (serum substitute) and keratinocyte growth factor (KGF). An in vitro skin equivalent model was used to examine the capacity of these cells to form an epidermis. Keratinocytes in different passages (P2, P4, and P6) and freshly isolated cells were studied. Keratinocytes grown on collagen type IV were able to form an epidermis at higher passage numbers than cells grown in the absence of collagen type IV (P4 and P2, respectively). In both cases the reconstructed epidermis showed an increased expression of Ki-67, SKALP, involucrin, and keratin 17 compared to normal skin. Only 50,000 keratinocytes grown on collagen type IV in P4 were needed to form 1 cm 2 epidermis, whereas 150,000 of freshly isolated keratinocytes were necessary. Using this culture technique sufficient numbers of keratinocytes, isolated from 1 cm 2 skin, were obtained to cover 400 cm 2 of wound surface in 2 weeks. The results show that keratinocytes can be cultured without the need of a fibroblast feeder layer and FCS and that these cells are still able to create a fully differentiated epidermis. This culture technique can be a valuable tool for the treatment of burn wounds and further development of tissue engineered skin.
Journal of Investigative Dermatology, 1993
This study compares two techniques for making cultured skin substitutes: a composite graft made of human fibroblasts and keratinocytes on a collagen-glycosaminoglycan membrane (CG) and a cultured epidermal cell sheet graft (CEG), without a dermal component. The "take" and quality of these cultured skin substitutes were evaluated by placing them on full-thickness, excised wounds of athymic mice. These cultured skin substitutes were placed onto 2-X-2em wounds created on athymic mice. Mice were sacrificed at days 10, 20, and 42 with histologic sections obtained for light, electron, immunofluorescent, a?d immunohistoche~ ical microscopy. "Take" was determmed separately by a dIrect immunofluorescent stain for human leukocyte ABC antigens. There were ten mice of each graft type with at least two animals sacrificed at each time point. Results showed positive "take" for all animals. Grossly, T he use of cultured skin substitutes in burn care and reconstructive surgery is expanding. Two key factors have shaped the design of these cultured grafts: the ability to grow keratinocytes in vitro and the inc~eas ing practice of early wound excision in the extensively burned patient. Medawar reported the first successful isolation of buman keratinocytes in 1941 [1], but it was not until 1975 that Rheinwald and Green reported the growth of human keratinocytes at clonal densities on a feeder layer [2]. This achievement, the culturing of a great number of epidermal cells with dividing times under 24 h, meant that enough keratinocytes could be grown in 3-4 weeks to cover a whole body surface area. Further work in this field over the last 15 years has led to decreased culture times and the formation of serum-free systems [3-6] that can match the growth of the previous serum-based systems. The second major step leading to more experimentation with permanent biologic wound coverings was the increased use of early excision of the burn wound with major burn victims. Several centers Manuscript
Burns, 2008
Transplantation of autologous cultured keratinocytes in single cell suspension is useful in the treatment of burns. The reduced time needed for culture, and the fact that keratinocytes in suspension can be transported from the laboratory to the patient in small vials, thus reducing the costs involved and be stored (frozen) in the clinic for transplantation when the wound surfaces are ready, makes it appealing. We found few published data in the literature about actual cell survival after transplantation of keratinocytes in single cell suspension and so did a comparative in vitro study, considering commonly used application techniques. Human primary keratinocytes were transplanted in vitro in a standard manner using different techniques. Keratinocytes were counted before and after transplantation, were subsequently allowed to proliferate, and counted again on days 4, 8, and 14 by vital staining. Cell survival varied, ranging from 47% to >90%, depending on the technique. However, the proliferation assays showed that the differences in numbers diminished after 8 days of culture. Our findings indicate that a great number of cells die during transplantation but that this effect is diminished if cells are allowed to proliferate in an optimal milieu. A burned patient's wounds cannot be regarded as the optimal milieu, and using less harsh methods of transplantation may increase the take rate and wound closing properties of autologous keratinocytes transplanted in a single cell suspension.
The Keio Journal of Medicine, 1990
An optimum culture condition was established for our keratinocyte cell culture system from human adult skin using 3T3 feeder cells. Calcium ion (Ca++) concentration was found to be critical and cells grew best at the Ca++ concentration of 0.2mM. Keratinocyte proliferation was promoted when 0.4ƒÊg/ml hydrocortisone and 7ƒÊg/ml insulin were added. However, epidermal growth factor, cholera toxin and transferrin did not show stimulating effects on multiplication of human keratinocytes in our culture system. The epidermal sheets grown in vitro under this optimized condition were transplanted onto athymic mice, and a short term of take was observed.
Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line
The Journal of Cell Biology, 1988
In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation-specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a p...
Transplantation of keratinocytes in the treatment of wounds
The American Journal of Surgery, 1995
Keratinocyte grafting can be used to treat acute traumatic and chronic non-healing wounds. The keratinocyte sheets are fragile and clinical "take" is difficult to assess, especially as activated keratinocytes secrete many growth factors, which have effects on wound healing apart from take. We have developed animal models of grafting that allow us to examine factors influencing autologous keratinocyte graft take. Results show clearly that pretreatment of the wound bed with viable dermis greatly increases the take of keratinocyte grafts. International literature. As a greater understanding of the complex interactions of cell and matrix evolve, so will potential therapeutic maneuvers, not just in the field of cultured keratinocyte grafts, but clearly in that of benign tumors, for example, keloids, and that of oncology. There is now overwhelming evidence of the requirement for a dermal substitute for cultured keratinocyte autografts, and the sheet complexity of the situation demands that this should approximate live human dermis as closely as possible. The stumbling blocks relate to avoiding the risks of viral transmission, tissue matching of host and donor, providing early epithelial cover, and improving delivery systems for fragile keratinocyte grafts.