Non-permeable Cryoprotectants' Influence on Fibroblast Slow Freezing in Six-banded Armadillo

BACKGROUND: There is a crucial need to develop appropriate cryopreservation solutions so that somatic resource biobanks of wildlife can be established. OBJECTIVE: Here, we propose a cryopreservation protocol to optimize the preservation of skin-derived fibroblasts from six-banded armadillos (Euphractus sexcinctus Linnaeus, 1758) by comparing different concentrations of fetal bovine serum (FBS) in the absence or presence of sucrose as non-permeable cryoprotectants. MATERIALS AND METHODS: Cells were cryopreserved by slow freezing with different solutions containing Dulbecco's modified Eagle's medium (DMEM) with 10% dimethyl sulfoxide (DMSO), varying concentrations of FBS (10, 20 and 40%) without or with 0.2 M sucrose, totaling six comparison groups. Cells not subjected to cryopreservation were used as a control. Cells were evaluated for morphological characteristics, viability, metabolism, apoptosis levels, proliferative activity and mitochondrial membrane potential (ΔΨ m). RESULTS: Cells maintained similar fusiform morphology and demonstrated high viability (> 90%) before and after cryopreservation in all groups. Cryopreserved cells with 10 and 40% of FBS without sucrose showed lower metabolism, but, when sucrose was added, this parameter was maintained as in the control group. This effect was not observed in the 20% FBS groups in the absence or presence of sucrose, with viability similar to that of the non-cryopreserved group. The addition of sucrose maintained apoptosis levels, while the 20 and 40% FBS without sucrose groups showed alterations in viable, early apoptosis and necrosis stages. Nevertheless, all cryopreserved groups showed lower proliferative activity with a higher population doubling time (16.2-19.9 h) than the non-cryopreserved group (15.2 h). Finally, the 20% FBS groups, in the absence or presence of sucrose, maintained the ΔΨm. CONCLUSION: We demonstrated that 20% FBS with sucrose was the most suitable cryopreservation solution for six-banded armadillo skin-derived fibroblast lines, promoting high cell survival after thawing.