Conditions for Culturing Biopsy Punches from Ngege (Oreochromis esculentus) Caudal Fins and Applications for Future Conservation Efforts.
Many factors are known to influence long-term growth potential of primary cell cultures, such as cell culture conditions and culture establishment techniques. This study investigates the relationship between fish cell culture conditions and cell viability as a means of developing cell culture procedures for the conservation of endangered freshwater fish species. The cell culture conditions investigated include: donor age, biopsy size, sampling location, enzymatic dissociation treatments and incubation temperature. Culture quality was assessed for cell viability, oxidative stress, senescence and cell death. The conditions that were most effective for culture quality were 6 mm biopsy punches taken from the proximal area of the caudal fin, digested with collagenase I for 60 minutes, and grown in L-15 medium containing 1x Primocin at 25oC. Viable cell concentrations obtained from fluorescent staining confirmed that cell populations declined to less than half their initial concentration within 7 days in culture. Autophagy and apoptosis may play a role in the resulting cell death inferred by the presence of vacuoles and lysosomal activity observed on advanced cultures. The presence of vacuoles, oxidative stress and senescence among treatment groups are indicative of the inadequacies in the presently evaluated protocols. Further research into the conditions required for culturing healthy cell lines is essential for fish somatic cell banking.