Assessing the resistance of sheep fibroblasts to increasing concentration of Trehalose

Trehalose is a non-reducing disaccharide commonly used as a cryo-protectant for deep freezing of cells. In this preliminary work, we have assayed the feasibility of its use for inducing reversible drying in somatic cells (fibroblasts), using sheep as a model. Here we have assayed the tolerance of somatic cells to increasing concentration of trehalose. To this extends, sheep fibroblasts were incubated using a growing concentration gradient of trehalose 50mM, 100mM, 250mM, 400mM and 500mM in Minimum Essential Medium (MEM) for 24hours in an incubator set at 38.5°C. On the basis of preliminary trials, the highest concentrations were excluded because they were found to be toxic to the cells and we have focused on the following: 50mM, 100mM and 200mM. The final osmolarity of the three concentrations were 376 mOsm/kg, 462 mOsm/kg and 711 mOsm/kg respectively. Cells have been incubated in MEM with trehalose for 3 hours in a stove set at 37°C and 12% humidity in lack of gas. Every hour cells were assayed for morphology and viability through Trypan Blue exclusion test. After one hour, live cells were about 71%, 63.15% and 52.85% respectively for 50mM, 100mM and 200mM explaining a negative correlation between the percentage of live cells and the sugar concentration. However, after 2 and 3 hours, death of cells was observed within each concentration as well as morphological changes which was however reversible upon washing cells from trehalose and further culture.