O-6: Cryopreservation and Long-Term Maintenance of Bovine Embryo-Derived Cell Lines

Background: The aim of this study was to develop methods for cryopreservation and long-term maintenance of putative bovine embryonic stem cells (ESCs). Materials and Methods: Putative bovine ESC (bESC) lines (n=3) isolated in conventional medium were used to compare slow-freezing and vitrification. Results: After warming, vitrified cells (96.9%) demonstrated significantly (p<0.05) better survival than frozen-thawed cells (81.5%) and formed significantly more colonies with good morphology (vitrification: 93/93, 100.0%, slow-freezing: 74/106, 69.81%, p<0.05). The effect of inhibitors of differentiation (PD184352, SU5402, CHIR99021) on ESC maintenance was assessed on putative bESC lines established in N2B27-3i medium (n=8) or conventional medium (n=1) after culture over 30 passages (>240 days). All cell lines expressed ALP, SSEA1, SSEA4, OCT4, REX1 and SSEA1. OCT4 expression was confirmed by relative real-time PCR and was upregulated in early passages of putative bESCs cultured in N2B27-3i (2.9 ± 89-fold higher at Passage (P) 2-4), whereas the converse was observed later (P22-26; 2.2 ± 0.1- fold increase in conventional medium). Putative bESC lines isolated in N2B27-3i medium (n=3) or conventional medium (n=1) were vitrified at P18 and, after warming, were cultured for a further 12 passages. These cells survived vitrification and expressed OCT4, REX1, SSEA1, ALP, SSEA1 and SSEA4. Conclusion: These results demonstrate that putative bESC lines that express pluripotent markers can be cultured long term and retain expression of pluripotent markers after vitrification.