Cryopreservation of keratinocytes in a monolayer

The cryopreservation of cells in tissues is one of the major challenges in current cryobiology, especially with regard to the progressively increasing held of tissue engineering. It is very questionable whether protocols whic h were developed for the cryopreservation of isolated cells are also applic able for cells in more complex structures, such as tissues. As a starting p oint toward cryopreservation of these three-dimensional structures, the aim of this study was to find an optimum cryopreservation protocol for keratin ocytes in a monolayer (two-dimensional structure). These epidermal cells ca n be transplanted as a monolayer grown on an appropriate matrix for the tre atment of deep-dermal bums and leg ulcers. The successful cryopreservation of such transplants would offer the advantage of long-term storage and imme diate availability of the transplant. In our study, the variables investiga ted were the cryoprotective solution and the cooling rate. In order to find a nontoxic cryoprotective agent (CPA) which could be transplanted without an additional washing step, we included hydroxyethyl starch (HES) as a poss ible CPA in our experimental protocol with the commonly used CPAs Me2SO, gl ycerol, and ethylene glycol. For the evaluation, the cell survival rate was determined by dye exclusion (trypan blue) and the cell metabolism was inve stigated by cell activity assay (alamarBlue). In conclusion, the cryopreser vation protocol with 10 wt.-% HES resulted not only in the highest survival rate (72%) but also in thr highest metabolic activity of the cells after t hawing: comparable values for the other CPAs were: Me,SO, 48%; glycerol, 8% ; and ethylene glycol, 10%, (C) 1999 Academic Press.