In vitro cell-toxicity screening as an alternative animal model for coral toxicology: effects of heat stress, sulfide, rotenone, cyanide, and cuprous oxide on cell viability and mitochondrial function

Abstract

The logistics involved in obtaining and maintaining large numbers of corals hampers research on the toxicological effects of environmental contaminants for this ecologically and economically important taxon. A method for creating and culturing single-cell suspensions of viable coral cells was developed. Cell segregation/separation was based on specific cell densities and resulting cell cultures were viable for at least 2 mos. Low-density cells lacking symbiotic zooxanthallae and rich in mitochondria were isolated and cultured for toxicity studies. Cells were exposed to differing degrees or concentrations of heat stress, rotenone, cyanide, sulfide, and cuprous oxide. Cells were assayed for mitochondrial membrane potential using the fluorescent probe, JC-9, and for overall viability using the MTT/formazan spectrophotometric viability assay. Significant differences were observed between controls and treatments and the efficacy of this method was validated; only 2 cm(2) of tissue was required for a seven-point concentration-exposure series.

Scleractinian coral cell proliferation is reduced in primary culture of suspended multicellular aggregates compared to polyps

“…Enzyme digestion induces dissociation of individual cells and small cell clusters, which remain suspended or adhere to the culture substrate and may aggregate over time (Gates and Muscatine 1992;Frank et al 1994;Helman et al 2008;Downs et al 2010). The mesoglea gel separating the two epithelial layers (epiderm and gastroderm) of cnidaria is an extracellular matrix (ECM) which, in Hydra, has been reported to contain collagen type IV (Fowler et al 2000) associated to laminin in the subepithelial basal lamina and to fibrillar collagen type I (Shimizu et al 2008;Sarras 2012).…”

“…Alternately, coral soft tissue is chemically induced to detach, in response to removal of divalent cations (Gates and Muscatine 1992;Frank et al 1994;Kopecky and Ostrander 1999;Domart-Coulon et al 2001, 2004aMass et al 2012) or exposure to a reducing agent, such as N-acetylcysteine (Peng et al 2008). Enzymatic digestion has also been widely used to dissociate the tissue into single cells (Gates and Muscatine 1992;Frank et al 1994;Helman et al 2008;Downs et al 2010). Types and brand of enzymes used in dissociation studies vary between laboratories, and are often not detailed by authors, making it difficult to standardize protocols for coral cell isolation.…”

“…These methods yield either tissue (explant) cultures, or mixed cell type single dissociated cultures, which may be further enriched in specific cell types and for example applied to ecotoxicology testing (Downs et al 2010). Suspended multicellular isolates (explants) are formed in cell culture medium within 3 days of culture initiation after divalent cation removal (Kopecky and Ostrander 1999;DomartCoulon et al 2001DomartCoulon et al , 2004a.…”

Ecological Risk Assessment of Munitions Compounds on Coral and Coral Reef Health

“…Once tissue disaggregation occurred, the cell slurry was then subjected to two differential centrifugations using a swinging bucket rotor at 50 x g for 5 min. The cell slurry was then layered onto a Percoll ® step-gradient (as in Downs et al 2010;2013), and centrifuged at approximately 200 x g for 4 min (not shown). Different fractions of non-zooxanthella (term used for dinoflagellate symbiont, Symbiodinium sp.)…”

“…Calicoblasts were identified by having the highest intensity of red fluorescence; the largest number of mitochondria per cell (Davy et al 2012). Cells were cultured in a modified tissue culture media for 48 h (Downs et al 2010;2013). Coral cellculture media (pH 8.2) consisting of 1 mL RPMI-1640, 100x vitamin solution to 99 mL of SSS-ASW (36 ppt salinity), 50 mM Hepes/KOH (pH 8.2), 1 mM calcium chloride, 1 mM sodium pyruvate, 0.075 g/L D-glucose, 0.3 g/L galactose, 0.25 g/L sodium DL-lactate, 0.25 mM ascorbate, 0.05 g/L α-lipoic acid, 0.5 mM proline, 0.5 mM cysteine, 0.5 mM methionine, 0.01 mM hydroxycobalamin, 0.001 mM sodium folate, 1 g/L bovine albumin (V), 0.05 g/L succinate, and 0.25 g/L L-glutamine.…”