The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin

Background. Cell therapy is an emerging therapeutic strategy aimed at repla cing or repairing severely damaged tissues with cultured cells. Epidermal r egeneration obtained with autologous cultured keratinocytes (cultured autog rafts) can be life-saving for patients suffering from massive full-thicknes s burns, However, the widespread use of cultured autografts has been hamper ed by poor clinical results that have been consistently reported by differe nt burn units, even when cells were applied on properly prepared wound beds . This might arise from the depletion of epidermal stem cells (holoclones) in culture, Depletion of holoclones can occur because of (i) incorrect cult ure conditions, (ii) environmental damage of the exposed basal layer of cul tured grafts, or (iii) use of new substrates or culture technologies not pr etested for holoclone preservation. The aim of this study was to show that, if new keratinocyte culture technologies and/or "delivery systems" are pro posed, a careful evaluation of epidermal stem cell preservation is essentia l for the clinical performance of this life-saving technology. Methods. Fibrin was chosen as a potential substrate for keratinocyte cultiv ation. Stem cells were monitored by clonal analysis using the culture syste m originally described by Rheinwald and Green as a reference. Massive full- thickness burns were treated with the composite allodermis/cultured autogra ft technique. Results. We show that: (i) the relative percentage of holoclones, meroclone s, and paraclones is maintained when keratinocytes are cultivated on fibrin , proving that fibrin does not induce clonal conversion and consequent loss of epidermal stem cells; (ii) the clonogenic ability, growth rate, and lon g-term proliferative potential are not affected by the new culture system; (iii) when fibrin-cultured autografts bearing stem cells are applied on mas sive full-thickness burns, the "take" of keratinocytes is high, reproducibl e, and permanent; and (iv) fibrin allows a significant reduction of the cos t of cultured autografts and eliminates problems related to their handling and transportation. Conclusion. Our data demonstrate that: (i) cultured autografts bearing stem cells can indeed rapidly and permanently cover a large body surface; and ( ii) fibrin is a suitable substrate for keratinocyte cultivation and transpl antation. These data lend strength to the concept that the success of cell therapy at a clinical level requires cultivation and transplantation of stem cells. W e therefore suggest that the proposal of a culture system aimed at the repl acement of any severely damaged self-renewing tissue should be preceded by a careful evaluation of its stem cell population.