Abstract
Marine mammal cell cultures are a multifunctional instrument for acquiring knowledge about life in the world’s oceans in physiological, biochemical, genetic, and ecotoxicological aspects. We succeeded in isolation, cultivation, and characterization of skin fibroblast cultures from five marine mammal species. The cells of the spotted seal (Phoca largha), the sea lion (Eumetopias jubatus), and the walrus (Odobenus rosmarus) are unpretentious to the isolation procedure. The sea otter (Enhydra lutris) fibroblasts should be isolated by trypsin disaggregation, while only mechanical disaggregation was suitable for the beluga whale (Delphinapterus leucas) cells. The cell growth parameters have been determined allowing us to find the optimal seeding density for continuous and effective cultivation. The effects of nonpathogenic algal extracts on proliferation, viability, and functional activity of marine mammal cells in vitro have been presented and discussed for the first time.
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Acknowledgments
The authors express their sincere gratitude to Professor Nelly A. Odintsova for the valuable discussion and support, and to Dr. Igor Manzhulo for providing vimentin antibodies. The study was partly conducted in the Center for Collective Use “Primorsky Aquarium” FEB RAS.
Funding
This research was supported by the grant of the President of Russian Federation for young scientists № МК-264.2017.4 and the grant of the Russian Foundation of Basic Research № 19-04-00752.
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AVB contributed substantially to the study’s conceptualization, marine mammal fibroblast isolation and cultivation, cytometrical analysis, cytotoxicity testing, and to the original manuscript preparation. YOK contributed to the study’s conceptualization, the fibroblast cultivation, data acquisition, and to the preparation of the manuscript. RVG contributed to the fibroblast cultivation and data acquisition. OGS and MAS contributed to the study’s conceptualization, the isolation and cultivation of algal cells, and the preparation of the manuscript. KVE contributed to molecular-genetic authentication of marine mammal fibroblasts and the preparation of the manuscript. IOK contributed to taking care of marine mammals and assisting in the biopsy procedure. MAM contributed to the study’s conceptualization, fibroblast cultivation, data acquisition, and immunocytochemical staining and observation, and to the preparation of the manuscript. All authors have reviewed and approved the final submitted manuscript.
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Appendix 1
Figure 8 Algorithm of flow cytometry analysis of marine mammal skin fibroblasts. Excluding cell aggregates by gating single events on FSC-A against FSC-H graph (a). Excluding cell debris and choosing the population of interest (“analyzed cells”) (b). Gating live (DAPI-negative) and dead (DAPI-positive) cells (c). Gating flow cytometry size calibration particles (# F13838, Molecular Probes) (d). Using gates from the size calibration particles for assessing a distribution of fibroblasts by their sizes (e). (PNG 2033 kb)
Appendix 2
Nucleotide sequences of control region mtDNA including tRNA-Thr and tRNA-Pro gene sequence for marine mammal cell cultures. (TXT 4 kb)
Appendix 3
Nucleotide sequences of ND2 gene mtDNA for marine mammal cell cultures. (TXT 6 kb)
Appendix 4
Amino acid sequences of ND2 gene mtDNA for marine mammal cell cultures. (TXT 2 kb)
Appendix 5
Nucleotide sequences of COI gene mtDNA for marine mammal cell cultures. (TXT 4 kb)
Appendix 6
Figure 9 Parameters of fibroblast of the common bottlenose dolphin Tursiops truncatus in culture: flow cytometry 2D plots of cells cultivated in the medium supplemented with 10% FBS (a) or 30% FBS (b), cell size distribution (c), the growth curves of cells cultivated in the medium supplemented with 10% FBS (d) or 30% FBS (e). (PNG 1913 kb)
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Boroda, A.V., Kipryushina, Y.O., Golochvastova, R.V. et al. Isolation, characterization, and ecotoxicological application of marine mammal skin fibroblast cultures. In Vitro Cell.Dev.Biol.-Animal 56, 744–759 (2020). https://doi.org/10.1007/s11626-020-00506-w
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DOI: https://doi.org/10.1007/s11626-020-00506-w