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  • Selection of an optimal culture medium and the most responsive viability assay to assess AgNPs toxicity with primary cultures of Eisenia fetida coelomocytes.

Selection of an optimal culture medium and the most responsive viability assay to assess AgNPs toxicity with primary cultures of Eisenia fetida coelomocytes.

Ecotoxicology and environmental safety (2019-08-26)
N Garcia-Velasco, A Irizar, E Urionabarrenetxea, J J Scott-Fordsmand, M Soto
ABSTRACT

Earthworm immune cells (coelomocytes) have become a target system in ecotoxicology due to their sensitivity against a wide range of pollutants, including silver nanoparticles (AgNPs). Presently, in vitro approaches (viability assays in microplate, flow cytometry, cell sorting) with primary cultures of Eisenia fetida coelomocytes have been successfully used to test the toxicity and the dissimilar response of cell subpopulations (amoebocytes and eleocytes) after PVP-PEI coated AgNPs and AgNO3 exposures. In order to obtain reliable data and to accurately assess toxicity with coelomocytes, first an optimal culture medium and the most responsive assay were determined. AgNPs posed a gradual decrease in coelomocytes viability, establishing the LC50 value in RPMI-1640 medium at 6 mg/l and discarding that the observed cytotoxicity was attributable to its coating agent PVP-PEI. Exposure to AgNPs caused selective cytotoxicity in amoebocytes, which correlated with the Ag concentrations measured in sorted amoebocytes and reinforced the idea of dissimilar sensitivities among amoebocytes and eleocytes. Silver nano and ionic forms exerted similar toxicity in coelomocytes. The in vitro approaches with coelomocytes of E. fetida performed in this study have the capacity to predict impairments caused by pollutants at longer exposure levels and thus, provide rapid and valuable information for eco(nano)toxicology.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
RPMI-1640 Medium, Modified, with 20 mM HEPES and L-glutamine, without sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Polyvinylpyrrolidone, average mol wt 10,000
Sigma-Aldrich
Basal Medium Eagle, With Earle′s salts and L-glutamine, without sodium bicarbonate, powder, suitable for cell culture
Sigma-Aldrich
L-15 Medium (Leibovitz), With L-glutamine, powder, suitable for cell culture