Establishment and characterization of fibroblast cell lines from the skin of the Yangtze finless porpoise

Abstract

The Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis), as the sole freshwater subspecies of N. phocaenoides, is endemic to the Yangtze River and its adjacent lakes. Its population has declined significantly over recent decades. In this study, we established a skin-derived finite fibroblast cell line of the Yangtze finless porpoise, named YFP-SF1, using primary cell culture methods, and an immortalized cell line, T-YFP-SF1, through co-transfection (GFP and SV40 T antigens) techniques. YFP-SF1 proliferated continuously with a minimum population doubling time of 31 h and exhibited age-dependent changes in growth rate. T-YFP-SF1 cells exhibited fibroblast morphology and were characterized by a shorter doubling time, higher attachment efficiencies, colony formation at a low seeding density, and growth in low serum concentrations. Anchorage independence and foci formation in the cell monolayer were observed from passage 36. The chromosome number of YFP-SF1 and T-YFP-SF1 remained stable at 2n=44 in the early passages, and the viability of thawed cells remained above 90% after cryopreservation in liquid nitrogen. Taken together, we have established fibroblast cell lines of Yangtze finless porpoise for the first time, which might assist as an in vitro model for this endangered mammal.

Establishment and characterization of pygmy killer whale (Feresa attenuata) dermal fibroblast cell line

“…In the present study, we developed dermal fibroblast cell line (PKW-LWH) and established fibroblast cell line (PKW-LWHT1) by transfecting SV40 T antigen gene into a primary cell line. A similar approach was applied to develop primary cell culture line from humpback whale skin tissue [ 15 ] and to establish the fibroblast cell line from Chinese white dolphin [ 16 ], and skin fibroblast from another cetacean species, Yangtze finless porpoise [ 11 ].…”

“…Similarly, Jin et al [ 16 ] showed 44 chromosomes in Chinese white dolphin fibroblast cells. Cetacean chromosomes numbers often have 42 to 44 and may appear metacentric, submetacentric, telocentric and sub-telocentric [ 11 ]. Thus, these cell cultures may be useful for assessing genotoxic agents and for chromosomal breakage analysis.…”

“…Meanwhile, the growth of cryopreserved cells was similar to the subculture and growth curve was also noted parallel. Another study showed that cryopreservation was applied from early passages and revival manifested 90% cells recovery after cryopreservation [ 11 ]. The cryopreservation method by Stacey and Masters [ 21 ], demonstrated that long-term backup of cells and/or genetical material is required to protect the cells from morphological changes, genetical variation, and possible contaminations.…”

“…However, cells culturing and establishing primary and fibroblast cell lines can provide a unique opportunity for marine conservation research, estimation of mammalian biological responses, underlying molecular mechanisms and indeed animal cloning [ 9 ]. Furthermore, cultured cells and cell lines can be used for conservation of genetic resource in the laboratories [ 11 ]. Besides, environmental and pathological effects studies on marine mammals are also possible using cell culturing and model development, thus extending to toxicological, bacteriological, virological and epidemiological studies [ 12 ].…”

Lifetime extension of humpback whale skin fibroblasts and their response to lipopolysaccharide (LPS) and a mixture of polychlorinated biphenyls (Aroclor)

“…Over-expression of the SV40T and TERT target proteins has been performed in a wide range of mammalian cell lines; however, only a few studies report on SV40T-associated long-term cultivation of cetacean-derived cell cultures (e.g. epithelial cells from bottlenose dolphin ( Tursiops truncatus ) (Pine et al 2004; Yu et al 2005), humpback dolphin ( Sousa plumbea ) fibroblasts (Jin et al 2013) and Yangtze finless porpoise ( Neophocaena phocaenoides ) fibroblasts (Wang et al 2011). No previous study has reported on TERT transfection of cells from a marine mammal.…”