Isolation, Culture, and Differentiation of Blastema Cells from the Regenerating Caudal Fin of Zebrafish

The caudal fin of teleost fish has become an excellent system for investigating the mechanisms of epimorphic regeneration. Upon amputation of the caudal fin, a mass of undifferentiated cells, called blastema, proliferate beneath the wound-epidermis and differentiate into various cell types to faithfully restore the missing fin structures. Here we describe a protocol that can be used to isolate and culture blastema cells from zebrafish. Primary cultures were initiated from 36 h post-amputation (hpa) blastema and optimal cell growth was achieved using L-15 medium supplemented with 5% fetal bovine serum in plates either coated with fibronectin or uncoated. After seeding, zebrafish blastema cells formed a uniform culture and exhibited polygonal shapes with prominent nucleus, while various cell types were also observed after few days in culture indicating cell differentiation. Upon treatment with all-trans retinoic acid, zebrafish blastema cells differentiated into neuron-like and oligodendritic-like cells. Immunocytochemistry data also revealed the presence of mesenchymal and neuronal cells. The availability of blastema cell cultures could contribute to a better understanding of epimorphic regeneration by providing a mean to investigate the mechanisms underlying blastema cell differentiation. Furthermore, this protocol is simple, rapid, and cost-efficient, and can be virtually applied to the development of any fish blastema cell culture.