190 A PROCEDURE TO OBTAIN FIBROBLASTS FROM WILD ANIMALS

Abstract

The number of wild animals threatened or at risk of extinction constantly increases and, as a result, biodiversity decreases. Therefore, it is necessary to set in motion proceedings to preserve the genetic variability and to overcome factors leading to this situation. Banks of cryopreserved cells, such as skin fibroblasts, from wild animals could be used both for genetic studies and as a cell source for technologies able to reproduce individuals. In this study we report that a single basic protocol provides a method for obtaining, culturing, and cryopreserving skin fibroblasts from a wide range of wild animals. Skin biopsies were taken in Cantabria (Spain) from live, anesthetized brown bear (Ursus arctos, n = 4), and in Asturias (Spain) from carcasses of accidentally killed individuals from the following species: grey wolf (Canis lupus, n = 1), red fox (Vulpes vulpes, n = 1), eurasian badger (Meles meles, n = 1), ermine (Mustela erminea, n = 1), fallow deer (Dama dama, n = 7), pyrenean chamois (Rupicapra pyrenaica, n = 1), western roe deer (Capreolus capreolus, n = 1), wild boar (Sus scrofa, n = 1), striped dolphin (Stenella coeruleoalba, n = 1) and one avian species, capercaillie (Tetrao urogallus, n = 1). Once obtained and transported to the laboratory, all samples were processed identically. Tissue samples were manually cut into small pieces and enzymatically digested with 0.05% trypsin-EDTA (Sigma-Aldrich, Madrid, Spain) for 30 min, and then with collagenase (300 units/mL) (Sigma) for 14 h at 38°C. Disaggregated cells were centrifuged at 1600 rpm for 10 min and the pellet was diluted with D-MEM (Sigma) containing 10% FBS. Cells were counted and a minimum of 3 × 10⁵ cells were placed in a 25 cm² flask for culture under 5% CO₂ in air and high humidity at 37°C (red fox, badger, ermine, western roe deer) or 38°C (brown bear, grey wolf, fallow deer, pyrenean chamois, wild boar, striped dolphin, capercaillie). Confluent fibroblast monolayers were obtained after 5–7 days in culture except for the striped dolphin whose sample was contaminated. Two to three passages were performed using 75 cm² flasks before freezing fibroblasts in D-MEM containing 10% DMSO (Sigma) and 10% FBS. Total cell yield from a confluent flask was 5–11 × 10⁶ cells, depending on the animal species. Fibroblasts recovered from all thawed samples grew to confluence, and karyotypes were performed at this step. It was possible to obtain, culture, freeze, and thaw skin fibroblasts successfully from all the above-mentioned land species. Although more research is needed, preliminary data suggest that the above-described procedure can be applied to mammals and birds, thus helping to preserve genetic material of wild animals.

Grant Support: Consejería de Medio Ambiente, Ordenación del Territorios e Infraestructuras, Asturias, SPAIN. The authors acknowledge CEPESMA, Luarca, Asturias, SPAIN.