Isolation and culture of wapiti (Cervus elaphus) satellite cells

Abstract

Myogenic satellite cells (SC) were isolated from the sternomandibularis muscles of two, 227 kg, male wapiti (Cervus elaphus) and studied in primary cell culture. Wapiti-derived SC were capable of attaching to culture substrata and following the myogenic program of proliferation and differentiation to form multinucleated myotubes. Wapiti SC attached equally well to pig skin gelatin (PSG), fibronectin (FN), Matrigel® and plastic (P > 00.05), but cell viability measured at 120 h varied depending on initial substratum type. Pig skin gelatin (0.02% wt vol−1) was chosen for the majority of subsequent experimentation for cost efficiency. The greatest amount of wapiti SC proliferation was observed in media containing 10% (vol/vol−1) horse serum (HS), 15% HS and 15% fetal bovine serum (FBS) (P > 0.05). Wapiti SC proliferated more when exposed to HS and FBS than to sheep serum (SS) (P < 0.05). No proliferation, differentiation or decrease in cell viability was observed in Dulbecco's Modified Eagle Medium (DMEM) + 1% HS, DMEM + 2% HS, DMEM + 3% HS or DMEM + 4% HS (P > 0.05) after 120 h in vitro. Proliferation of SC was doubled when insulin was added to both 10% HS- and 2% HS-containing media (P < 0.05). Although insulin alone in serum-containing media did not promote fusion of wapiti SC, two defined media (ITT and ITT-CF) that contain insulin did promote fusion of wapiti SC cultures. ITT-CF induced 3% fusion of wapiti SC into myotubes, and ITT induced 1% (P < 0.05). There was also an increase in total cell numbers in SC exposed to ITT-CF in comparison with ITT, ovine defined media (ODM) or ovine defined media-Modified (ODM-Mod))(P < 0.05). Although defined media differed in their ability to induce proliferation or differentiation (P < 0.05), the substrata on which the SC were plated did not influence the defined media effect on SC activity (P > 0.05). Satellite cells exposed to ITT and ITT-CF differed morphologically from SC exposed to ODM and ODM-Mod, which may suggest that formulation differences are influencing wapiti-derived SC proliferation and differentiation. Key words: Wapiti, satellite cells, primary culture, myotubes