Optimization of media and transfection with SV40 large T antigen and c-myc for the establishment of an immortal sea urchin cell line

Immortal cell lines are cell cultures capable of indefinite growth without reaching senescence. Although many vertebrate and some terrestrial invertebrate cell lines have been produced, the establishment of an immortal marine invertebrate cell line remains an elusive goal. In general, marine invertebrate cell culture is greatly hindered by the lack of proliferation and long term viability of cultured cells. As such, much work in this field has focused on identifying media supplements and source cells amenable to long-term culture as well as optimizing immortalization methods. We surveyed seven media supplements (fetal bovine serum, newborn calf serum (NCS), horse serum, chicken serum, two commercial lipid mixtures and coelomic fluid) for their abilities to promote proliferation and long-term viability in culture. We also experimented with embryonic source tissues and developed an oncogene-mediated immortalization technique in hopes of producing transgenic marine invertebrate cells with greater proliferative capacity and decreased fastidiousness. We report that 1% supplementation of Leibowitz’s L-15 media with NCS or chicken serum afforded enhanced viability of coelomocytes as indicated by esterase activity and long term cell-to-substratum attachment compared to unsupplemented L-15 (statistical significance achieved for NCS; p<0.05, Duncan’s multiple range test). We also observed that embryonic tissue was capable of in vitro proliferation, but had significant contamination with protists, leading to rapid demise of cultures within 2 weeks. We observed the formation of colonies in coelomocyte cultures transfected with c-myc and SV40 large T antigen and report stable integration of the c-myc gene sequence in one cell culture five weeks after transfection; however, dramatically increased proliferation was not apparent over a 7 week period.