Abstract:This study aimed to investigate the methods of isolation, culture, and identification of melanophores and xanthophores in polychromatic midas cichlids (Amphilophus citrinellus) and to thus observe the growth characteristics of pigment cells in fish. In this study, the caudal fin, scales, and skin tissues of A. citrinellus were used as the experimental material. To separate the pigment cells, the tissues were digested in trypsin solution and collagenase solution for 6 h and 12 h, respectively, at 25℃~28℃. After the cell suspension was filtered through a stoma nylon net, xanthophores and melanocytes were separated and collected by means of 35%-45%-55% Percoll multilayer density gradient centrifugation. K-SFM medium was used for primary cell culture and cell purification to inhibit the growth of fibroblasts and keratinocytes. DMEM supplemented with phorbol ester (TPA), double resistance, and bEGF, etc., was used for the subculture of pigment cells. The morphology of the cells was observed by L-DOPA staining and transmission electron microscopy, and the cells were identified by molecular markers. The results showed that when the cells were separated, the melanocytes were located at the boundary of the 45% and 35% Percoll reagent layers, while xanthophores were located on top of the 35% Percoll layer. Both pigment cells condensed into flocculation. Transmission electron microscopy showed that the melanocytes contained a large number of melanosomes, while the yellow cells contained the MTX and carotenoid vesicles. The melanocytes were positive under L-DOPA staining. The 10th generation of pigment cells was used for PCR detection of the body color related genes MC1R, TYR, and EDNRB, which showed strong specificity of gene amplification bands, indicating that the two pigment cells obtained had good biological activity. In this study, methods for the isolation, culture, and identification of melanophores and xanthophores of polychromatic midas cichlids were successfully established, providing a cell model for further research on the molecular mechanisms of body color cell differentiation and body color formation in fish.
