Skin Graft Preservation

o. There are two different cryopreservation protocols used in skin preservation: slow freezing and quick freezing/snap frozen. Slow freezing can be achieved using a controlled-rate cooling apparatus (Blondet et al., 1982). There is an optimum cooling rate for any cell type that produces maximum cell survival. On either side of this optimum, the survival rate falls. In the presence of CPA, a cooling rate of -1 oC per minute will ensure survival of most of the cells within skin tissue. As the cooling rate is increased,cell populations are sequentially and adversely affected. Many cells in the body derived from leukocytes or closely related lineages are known to be exquisitely sensitive to cryogenic injury. The depletion of immunostimulatory “passenger leukocytes” was demonstrated by increasing the cooling rate for pancreatic islets of Langerhans while maintaining the viability of the insulinproducing islet cells (Ingham et al., 1993). This concept of cooling rate–dependent immunomodulation was evaluated for skin tissue. A cooling rate of -30襖/min was shown to maintain the viability of keratinocytes and fibroblasts while reducing the immunogenicity (as assessed by the mixed epidermal cell/lymphocyte response assay) of murine allografts by 95%. This was assumed to be due to an effect of the faster cooling rate on the major immunostimulatory cell in the skin—the Langerhans cell. Quick freezing/snap frozen is skin vitrification technique. Vitrification is defined as “the instant solidification of a solution brought about by an extreme elevation in viscosity during cooling, without ice crystal formation”. In other words, vitrification is faster and lacks some of the typical disadvantages seen in traditional slow freezing (Mukaida, 2003). It bypasses the ice-crystal formation phase and instantaneously solidifies into a glass-like structure, moves the water directly into a glass-like phase. In a glass, the molecules do not rearrange themselves into grainy ice crystals as the solution cools, but instead become locked together while still randomly arranged as in a fluid, forming a “solid liquid” as the temperature falls below the glass transition temperature (Silvestre et al., 2002). This technique was simple, and no expensive equipment was needed. Initially, this method needed a 2-step procedure because the cryoprotective agent was toxic. Kasai et al., (1990) modified this impractical to a 1-step method and incubated the skin with vitrification solution at room temperature and was transferred directly into liquid nitrogen.