Influence of Intracellular Cryoprotectants on the Conservation of Dermal Somatic Tissues Derived from Antillean Manatees (Trichechus manatus manatus Linnaeus, 1758).

Abstract

Cryopreservation of somatic tissue has been studied as a tool for the knowledge and conservation of endangered species, such as Antillean manatees. The use of vitrification protocols is an important step in the establishment of biological banks. To decrease the damage caused by this technique, a reduction in the concentration of cryoprotectants has been proposed. Therefore, we aimed to evaluate combinations and concentrations of intracellular cryoprotectants for the conservation of somatic tissues derived from Antillean manatees. Dulbecco's modified Eagle's medium, F-12 composed of 10% fetal bovine serum and 0.25 M sucrose, was supplemented with 3.0 M ethylene glycol (EG) plus 3.0 M dimethyl sulfoxide (DMSO), or 1.5 M EG plus 1.5 M DMSO or 3.0 M EG or 3.0 M DMSO, to produce four solutions for solid-surface vitrification. Noncryopreserved tissues were used as the controls. After warming, tissues derived from four Antillean manatees were evaluated for ultrastructure, histology, and in vitro culture. No differences were observed among the cryopreserved and noncryopreserved tissues in terms of ultrastructure. The dermis thickness of the cryopreserved fragments in solutions containing 3.0 M EG plus 3.0 M DMSO, 3.0 M EG, and 3.0 DMSO was similar to that of the control. Moreover, cryopreservation with 3.0 M EG plus 3.0 M DMSO maintained tissue proliferative capacity potential evaluated by quantification of nucleolar organizing regions. Nevertheless, none of the cryopreserved fragments were able to maintain the number of fibroblasts and the collagen percentage as compared with that of the noncryopreserved fragments. Also, none of the cryopreserved fragments in the different solutions were able to produce cells in vitro. In summary, even reducing the concentration of intracellular cryoprotectants as well as their association did not guarantee the maintenance of cells after in vitro culture. Further studies are needed to optimize the cryopreservation protocols in Antillean manatee somatic tissues.