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

• Main Authors: Parameswaran Vijayakumar, M. Leonor Cancela, Vincent Laizé
• Format: Article
• Language: English
• Published: MDPI AG 2020-01-01
• Series: Fishes
• Subjects: primary cell culture; blastema; zebrafish; fin regeneration; cell differentiation
• Online Access: https://www.mdpi.com/2410-3888/5/1/6

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-<i>trans</i> 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.