Characterization of goldfish fin cells in culture: some evidence of an epithelial cell profile.

Comprehensive characterization of cultured cells in fish was little explored and cell origin is often deduced from morphological analogies with either epithelial of fibroblastic cells. This study aims to characterize cell origin in goldfish fin culture using morphological, immunochemical, and molecular approaches. Time lapse analysis revealed that cultured cell morphology changed within minutes. Therefore, cell morphology cannot predict whether cells are from fibroblastic or epithelial origin. The labeling pattern of heterologous anti-cytokeratin and anti-vimentin antibodies against goldfish epithelial cells and fibroblasts was first tested on skin sections and the corresponding labeling of the cultured cells was analyzed. No cell origin specificity could be obtained with the chosen antibodies. In the molecular approach, detection levels of three cytokeratin (CauK8-IIS, CauK49-IE and CauK50-Ie) and one vimentin transcripts were assessed on skin and fin samples. Specificity for epithelial cells of the most abundant mRNA, CauK49-Ie, was thereafter validated on skin sections by in situ hybridization. The selected markers were used afterwards to characterize fin cultures. CauK49-IE riboprobe labeled every cell in young cultures whereas no labeling was observed in older cultures. Accordingly, CauK49-IE transcript levels decreased after 15 days culture while CauK8-IIS ones increased. The use of homologous marker gave evidence that young cultured cells from goldfish fin are homogeneously of epithelial type and that cell characteristics may change over culture time.

Cryobanking of fish somatic cells: optimizations of fin explant culture and fin cell cryopreservation.

When gametes or embryos are not available, somatic cells should be considered for fish genome cryobanking of valuable or endangered fish. The objective of this work was to develop a method for fin explant culture with an assessed reliability, and to assess fin cells ability to cryopreservation. Anal fins from goldfish (Carassius auratus) were minced and gently loosened with collagenase before explants were plated at 20 degrees C in L-15 medium supplemented with fetal bovine serum and pH buffering additives. Quantification of cell-donor explants per fin rated the culture success. Cells were successfully obtained from every cultured anal fin (mean = 65% cell-donor explant per fin). All other fin types were suitable except the dorsal fin. Explant plating could be deferred 3 days from fin collecting. Fins from seven other fish species were successfully cultured with the method. After 2-3 weeks, sub-confluent fin cells from goldfish were cryopreserved. Cryopreservation with dimethyl sulfoxide and sucrose at a slow freezing rate allowed the recovery of half the goldfish fin cells. Cells displayed the same viability as fresh ones. 1,2-propanediol was unsuitable when a fast freezing rate was used. The procedure could now be considered for cryobanking with only minimal adaptation to each new species.