From skin biopsies to primary fibroblast cultures: cytotoxicity assessment of environmental stressors on humpback whale

Details

Supervisors

Debier, Cathy

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en sciences agronomiques, à finalité spécialisée

Abstract

enCetaceans are increasingly exposed to multiple environmental and anthropogenic stressors, including persistent organic pollutants (POPs), physiological stress, and infectious diseases. Understanding the combined effects of these stressors remains challenging because controlled in vivo experiments are largely precluded by ethical, legal and logistical constraints. Consequently, the development of cetacean-specific in vitro models is essential to investigate cellular mechanisms underlying toxicological responses. The aim of this thesis was to establish primary fibroblast cultures from humpback whale (HW) (Megaptera novaeangliae) skin biopsies and evaluate their suitability for multiple-stressor toxicological studies. Fresh HW biopsies collected in Northern Norway were cultured using an explant-based approach to isolate fibroblasts, which were subsequently expanded and cryopreserved to establish a fibroblast biobank. In parallel, several protocols were tested to recover fibroblasts from cryopreserved HW and pilot whale (PW) (Globicephala melas) skin samples. Fibroblasts isolated from fresh HW biopsies were then exposed to POPs, cortisol (CORT), and lipopolysaccharide (LPS), applied individually and in combination. Cell viability was assessed using complementary CFDA, PrestoBlue and LDH assays. Among the 30 fresh HW biopsies processed, 13 successfully generated fibroblast cultures that were expanded and cryopreserved, resulting in the establishment of a fibroblast biobank. In contrast, fibroblast recovery from cryopreserved tissues was largely unsuccessful, with only one out of 17 cryopreserved samples showing fibroblast outgrowth and reaching partial confluence despite testing different culture conditions. Exposure experiments revealed a concentration-dependent cytotoxicity of POPs, with significant reductions in fibroblast viability observed from 50X environmental concentrations onwards. CORT induced marked cytotoxicity only at the highest concentration tested (1000 µM), whereas LPS had limited effects on viability. Co-exposure to POPs and CORT did not modify POP-induced cytotoxicity, while the combined presence of POPs, CORT and LPS resulted in lower EC50 values and an earlier onset of viability loss. Overall, this study demonstrates that primary fibroblasts can be successfully established from fresh HW skin biopsies and constitute a promising cetacean-specific in vitro model for toxicological investigations. The results also highlight the need for further optimization of cryopreservation procedures and provide dose-response relationships that will support future transcriptomic, proteomic and cytokine-based investigations of multiple-stressor effects in cetacean fibroblasts.