Bums (1993) 19, (5), 406-410 Printed in Great Britain Cryopreservation of cultured dermal fibroblast impregnated collagen gels B. Teasdale’, V. K. Siebe?, D. J. Riches1 and J. Nanchaha13 ‘Skin Biology Unit, Department of Anatomy, Queen Mary and Westfield College (University of London), 2Department of Life Sciences, University of East London and ‘St Lawrence Hospital, Plastic and Reconstructive Surgery Centre, Chepstow, Gwent, UK The survival of cultured a2rmal fibroblasfs was evaluated following manufacture, fieezkg and disaggregation of fibrobk&-imprepted col- lagen gels. The concentration which gave optimal rell survival was determined for three cyoprofecfanfs Cplycerol, dimefhyl sulphoxide (DMSO) and efhanediolj ana’ their efficacy compared. DMSO led to the highest cell viability after freezing atd thawing. The effect of rate of freezing was also compared and 0.5” Umin (within the range 20°C to - 70” Cj was found to result in a sign$canf enhancement of cell viability in comparison with freeu’ng at ~.o”C/min or rapid freezing. Introduction There is a considerable need to replace skin lost through injury, particularly bums, and the most common source is through split-thickness skin grafts. However, problems associated with donor sites include pain, fluid loss, and infection; healing of donor sites may be slow, particularly in the elderly, whilst hypertrophic scar formation may occur especially in children. When limited donor sites are available such as following extensive bums, grafts can be expanded by meshing techniques (Tanner et al., 1964) but greater than three- to four-fold expansion is associated with slow healing (Alexander et al., 198’1). Bum wounds may be temporarily covered with allogenic skin but there is a risk of infection, including hepatitis and HIV (Clarke, 1987) and, in addition, as calculated in the USA demand has outstripped supply by a factor of 5 to 7 (De Clement and May, 1984). Allografts are a very effective dressing and methods for the cryo- preservation of cadaver skin, in order to retain maximum viability, have been studied extensively (May et al., 1984). The problem of limited donor sites may be overcome by culturing keratinocytes in vitro, permitting up to a 10 OOO- fold expansion (Gallico et al., 1984). Under appropriate conditions, keratinocytes will differentiate and stratify (Green et al., 1979; Pitteklow and Scott, 1986). Sheets of cultured keratinocytes have been applied to burns (O’Conner et al., 1981; Gallico et al., 1985; Madden et al., 1986), ulcers (Leigh and Purkiss, 1986) and wounds follow- ing the excision of giant hairy naevi (Gallico et al., 1989) and tattoos (Brain et al., 1989). However, keratinocyte sheets do not correct contour defects, are prone to spontaneous blistering (Woodley et al., 1988; Desai et al., 1992) and tend to contract (Gallico et al. 1989). Allogenic keratinocyte sheets have been found to survive for less than 7 days when 0 1993 Butterworth-Heinema Ltd 0305-4179/ 93/ 050406-05 applied to full skin thickness defects (Burt et al., 1989; Brain et al., 1989; De Luca et al., 1989) but accelerated wound healing occurred, possibly due to the release of growth factors (Eisinger et al., 1988). Cultured keratinocytes may be used at centres distant from the source laboratory (De Luca et al., 1989) as they can readily be transported whilst frozen. Cultured cell suspen- sions are routinely stored for prolonged periods in liquid nitrogen (Freshney, 1986) and there is interest in the parameters which optimize cell viability. Keamey (1990) described a protocol which retained the highest viability for frozen suspensions of keratinocytes and in a comparison of the effects of cryopreservant and cooling rate on viability he found that 10 per cent DMSO and slow cooling ( - l” C/min in the range 4°C to - 70°C) gave the optimum results. The problems associated with the use of sheets of cultured keratinocytes alone has led to the development of cultured skin-equivalent materials with both dermal and epidermal components (Nanchahal and Ward, 1992; Arons et al., 1992) and a variety of cellular and acellular models have been developed. In its simplest form Yannas et al. (1980) used an acellular cross-linked collagen and glyco- saminoglycan dermis overlaid with a Silastic sheet. Hans- borough et al. (1989) used a more complex collagen- glycosaminoglycan cross-linked fibroblast-impregnated gel overlain with keratinocytes, whilst Bell et al. (1983) described a dermis of a collagen gel impregnated with fibroblasts overlaid with an epidermis of cultured keratino- cytes. In a study of the concentration of collagen and fibroblasts required for a dermal graft, Rowling et al. (1990) found that 7.0 x lo3 cells/ml in a 0.5 per cent solution of collagen produced the optimal gel in terms of shrinkage and handling properties. Disaggregation of the gel by col- lagenase was inversely proportional to the length of time of incubation as the fibroblasts reorganized and cross-linked the collagen during incubation. Allogenic fibroblast impreg- nated collagen gels overlain with keratinocytes have been used in a small clinical trial on patients undergoing tattoo excision (Nanchahal et al., 1989). Graft survival was good with acceptable cosmetic results, and correction of contour defects and histological examination showed that immature gels were extensively remodelled following grafting, cell survival was followed using probes for the y-chromosome (w. R. Otto et al., personal communication). Hull and Cool (1990) grafted cultured skin equivalents onto bums using a