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.

Assessing the resistance of sheep fibroblasts to increasing concentration of Trehalose

Yosra Ressaissi;Marta Czernik;Paola Toschi;Pasqualino Loi
2018-01-01

Abstract

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.
2018
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11575/100951
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