Developing methodologies for the isolation and in vitro culture of ovarian cells would be a significant breakthrough for germ cell transplantation in fish. Investigations were conducted to develop a protocol for isolating and in vitro culture of ovary-derived cells from Black Crappie (Pomoxis nigromaculatus) and White Crappie (P. annularis). Ovarian tissues were obtained from one-year-old Black and White Crappies. Five different digestive enzymes: 500U/mL Collagenase type I, 500U/mL Collagenase type IV, 0.05% Trypsin-EDTA, 0.25% Trypsin-EDTA (Gibco), and TrypLETM Express were evaluated for cell isolation. Isolated cells from the ovarian tissue were next cultured in two different concentrations (10%, 20%) of Fetal Bovine Serum (FBS) in Leibovitz’s L-15 growth media. In addition, four incubation temperatures (15, 20, 25, and 30 °C) were evaluated to determine optimal culture temperature for these ovarian cells.
The number of live cells obtained from the 0.25% Trypsin-EDTA and TrypLETM Express treatments were significantly higher than other treatments. No significant difference in cell growth was observed between the 10% and 20% FBS treatments. Cell growth and division was seen at all incubation temperatures. Cells isolated using 0.25% Trypsin and TrypLE TM reached 80-90% confluency in 12.5 cm2 cell culture flask within five days of inoculation in 20, 25, and 30 °C incubation regimes. Whereas at 15°C incubation temperature, the cells took ten days post-inoculation to reach 80-90% confluency. Upon microscopy inspection, cells incubated at 20 and 25 °C appeared morphologically healthier than cells incubated at 30°C, where cell detachment from the substrate and irregular cell shape was observed. Cells were sub-cultured up to passage 2. Based on these findings, we conclude that 0.25% Trypsin and TrypLETM enzymes are optimal for cell disassociation and isolation, while an incubation temperature of 20-25 °C is favorable for primary cell culture in L-15 media supplemented with either 10 or 20% FBS.