When Simplicity Matters: Simplified Skin Tissue Cryopreservation Media for Wildlife Conservation

The conservation of threatened species requires effective strategies to preserve genetic diversity, with genome resource banks representing a valuable tool in this context. However, many cryopreservation protocols rely on complex media and laboratory infrastructure, limiting their applicability under field conditions.

This study evaluated whether simplified cryopreservation media composed of widely available reagents could effectively preserve skin tissue from Neotropical deer using slow freezing and needle-immersed vitrification.

Skin samples from red brocket deer (Mazama rufa) and marsh deer (Blastocerus dichotomus) were cryopreserved using conventional media (Ham's F10 with 10% fetal bovine serum, 0.5 M sucrose and 2.8 M dimethyl sulfoxide) or simplified saline-based formulations containing 0.5 M commercial table sugar and 2.8 M dimethyl sulfoxide. Post-thaw viability, mitochondrial activity, histomorphology, apoptosis, and fibroblast culture were evaluated.

Fibroblast cultures were successfully established from fresh and cryopreserved skin tissues, with cell migration occurring within 2-5 days and 70% confluence reached between 13 and 19 days depending on protocol. In red brocket, slow freezing showed higher post-thaw cell viability (79.2 ± 4.96% and 80.7 ± 8.26%) than vitrification (63.0 ± 12.3% and 59.2 ± 10.6%; p = 3.03 × 10^-5), but lower mitochondrial activity (69.5 ± 10.5%) compared with needle-immersed vitrification, which showed lower viability (59%-63%) but higher mitochondrial activity (90.1 ± 8.9% and 94.1 ± 5.2%; p = 0.00208). In marsh deer, viability differed significantly among protocols (p = 0.0194), with fresh tissue showing higher viability than conventional and simplified vitrification, while mitochondrial activity showed no significant differences (p = 0.9885). Simplified media performed comparably to conventional formulations across both cryopreservation techniques. Species-specific responses were evident, with marsh deer tissues showing similar responses across cryopreservation strategies, whereas red brocket exhibited greater sensitivity to vitrification.

The adoption of accessible and field-adapted cryopreservation strategies may facilitate implementation of somatic cell biobanking.