Isolation, in vitro culture, and characterization of black crappie, Pomoxis nigromaculatus and white crappie, P. annularis ovarian tissue primary cells

Abstract

Abstract Investigations were conducted to develop a general protocol for the isolation and in vitro culture of ovary‐derived cells from black crappie, Pomoxis nigromaculatus, and white crappie, P. annularis. Five digestive enzymes: 500 U/mL collagenase type I, 500 U/mL collagenase type IV, 0.05% trypsin‐EDTA, 0.25% trypsin‐EDTA, and 1X TrypLE™ Express were evaluated for live cell isolation. The isolated cells were cultured in 10 or 20% fetal bovine serum (FBS) in L‐15 growth media. In addition, four incubation temperatures (15°C, 20°C, 25°C, and 30°C) were also evaluated. The number of live cells obtained from the 0.25% trypsin and TrypLE™ Express treatments was significantly higher than other treatments. No difference in cell growth was observed between the two FBS treatments. Cells isolated using TrypLE™ Express and 0.25% trypsin reached 80% to 90% confluency in 12.5 cm2 cell culture flasks within 5 days of inoculation at 20 and 25°C. At 15°C, 10 days were required to reach 80%–90% confluency. Morphologically, cells incubated at 15°C, 20°C, and 25°C appeared healthier than cells incubated at 30°C, where irregular cell shape and substrate detachment were observed. We concluded TrypLE™ Express and 0.25% trypsin were optimal for cell dissociation and isolation of crappie ovarian cells. An incubation temperature of 20°C–25°C was favorable for cell culture in L‐15 media supplemented with either 10% or 20% FBS.

Keywords

• crappie
• incubation temperature
• ovarian cell isolation
• primary cell culture
• TrypLE™ express