Establishment, Characterization, and Toxicological Application of Loggerhead Sea Turtle (Caretta caretta) Primary Skin Fibroblast Cell Cultures

Abstract

Pollution is a well-known threat to sea turtles but its impact is poorly understood. In vitro toxicity testing presents a promising avenue to assess and monitor the effects of environmental pollutants in these animals within the legal constraints of their endangered status. Reptilian cell cultures are rare and, in sea turtles, largely derived from animals affected by tumors. Here we describe the full characterization of primary skin fibroblast cell cultures derived from biopsies of multiple healthy loggerhead sea turtles (Caretta caretta), and the subsequent optimization of traditional in vitro toxicity assays to reptilian cells. Characterization included validating fibroblast cells by morphology and immunocytochemistry, and optimizing culture conditions by use of growth curve assays with a fractional factorial experimental design. Two cell viability assays, MTT and lactate dehydrogenase (LDH), and an assay measuring cytochrome P4501A (CYP1A) expression by quantitative PCR were optimized in the characterized cells. MTT and LDH assays confirmed cytotoxicity of perfluorooctanoic acid at 500 μM following 72 and 96 h exposures while CYP1A5 induction was detected after 72 h exposure to 0.1-10 μM benzo[a]pyrene. This research demonstrates the validity of in vitro toxicity testing in sea turtles and highlights the need to optimize mammalian assays to reptilian cells.

Establishment and characterization of a cell line from the Chinese soft-shelled turtle Pelodiscus sinensis

“…The immunocytochemistry results in the present study showed the cells reacted strongly to the antibodies against vimentin, the specific marker for fibroblasts, while the cells reacted negative to the antibodies against cytokeratin, the epithelial marker, indicating PSF cells were fibroblastic. It was reported first as identification of fibroblast-like cells in P. sinensis and similar findings in a primary skin fibroblast cell of the sea turtle Caretta caretta have been described (Webb et al 2014). Isoenzyme analysis in this study revealed the PSF cell line had an LDH pattern of five bands and an MDH profile of two bands, which differs from other turtles or fish species.…”

“…Chromosome analysis, isoenzyme comparison, molecular biology, and immunohistochemistry techniques are commonly used for this purpose (Li et al 2009a, b;Freshney 2010;Wang et al 2013;Webb et al 2014). Karyotype analysis revealed a diploid chromosomal count of 2 N = 66 in majority of cells, which was identical to the modal chromosome number of PSF cell line; nevertheless, a variable karyotype formula was observed in P. sinensis cells because of the presence of sex chromosomes and the composition of sub-mediocentrics and relative small chromosome (Fu et al 2013).…”

“…Culture of a virus with suitable cell lines for further characterization of the virus and the development of efficient vaccines is essential to protect the animals from viral infections. Several continuous cell lines or cell cultures from turtles have been reported, including a cell line from the heart of the box turtle Terrapene carolina (Clark and Karzon 1967), an epithelial skin cell line derived from the green sea turtle (Koment and Haines 1982), primary fibroblast cultures from the hawksbill sea turtle (Fukuda et al 2012), melanomacrophage cultures from the liver and spleen of three turtle species representing three divergent families (Rund et al 1998), and a primary skin fibroblast cell line of the sea turtle Caretta caretta (Webb et al 2014). For Chinese soft-shelled turtle, a primary embryo cell culture was developed to study a poxvirus-like virus (Liu et al 2012) and a continuous cell line from the extracranial carotid artery was developed to study an iridovirus (Fu et al 2013).…”

Are we working towards global research priorities for management and conservation of sea turtles?

“…More recently, in vitro, or cell-based, approaches have been used as an ethical alternative for assessing the effects of contaminants in sea turtles (e.g. Wang et al 2013b, Webb et al 2014, Wise et al 2014, Young et al 2015. Investigating toxicity thresholds for pollutants and the effects of complex chemical mixtures are important areas for future research.…”

The current state and future directions of marine turtle toxicology research

“…The most common endpoint examined for in vitro exposure was cytotoxicity (n = 6), with 83% of these examining cytotoxicity of metals (Tan et al, 2010;Wang et al, 2013a;Wise et al, 2014;Young et al, 2015;Tong et al, 2016), and 17% examining organics (Webb et al, 2014). Two of these publications also examined genotoxicity (Wise et al, 2014;Young et al, 2015).…”