Effects of Temperatures and Basal Media on Primary Culture of the Blastomeres Derived from the Embryos at Blastula Stage in Marine Medaka Oryzias dancena

Abstract

Although the efforts to establish fish embryonic stem cells (ESCs) have been made for a long time, derivation of authentic ESCs that possess pluripotency is still difficult suggesting a need for the stepwise optimization of the methods to establish fish ESCs. Primary culture of the blastomeres from the embryos at blastula stage is a critical step for establishing continuous ESC lines. Here, we evaluated the effects of temperatures and basal media on primary culture of blastula embryo-derived blastomeres in marine medaka (Oryzias dancena). The blastomeres were isolated from the blastula embryos and cultured in various conditions designed by the combination of 4 temperatures including 28°C, 31°C, 34°C, and 37°C and 2 basal media including Dulbecco’s modified eagle’s medium (DMEM) and Leibovitz’s L-15 medium (L15). With the exception of a case cultured in L15 at 31°C, the rate of primary cell adherence reached 100% when the blastomeres were cultured over 31°C. The period for primary adherence was significantly shorter in the groups cultured in 34°C and 37°C than in the ones in 28°C and 31°C. The proportion of subculture was significantly high in the group cultured in DMEM at 31°C compared to the other groups. Collectively, we demonstrated that the culture in DMEM at 31°C was effective to primary culture of the blastomeres derived from blastula embryos.

Keywords

• blastomere
• blastula embryo
• primary culture
• temperature
• media