Abstract
Anthropogenic influences on the environment have been become a focal point for many social and political endeavors. With an ever-increasing rate of new contaminants being introduced into the environment every year, regulatory policies have begun to shift to prevention rather than mitigation. However, current in vivo testing strategies, in addition to ethical considerations, are too expensive and time consuming to adequately screen potential contaminants within a realistic timeframe. As a result, in vitro testing on cell cultures has been identified as an ideal alternative testing strategy for emerging contaminants. In the context of ecotoxicology, in vitro testing has had limited use particularly with marine invertebrates like the marine mussel Mytilus edulis mainly due to difficulties in establishing longer term cell cultures and cell lines. The aim of this study was to define an optimal technique (extraction and maintenance) for establishing a primary cell culture on M. edulis hemocytes that could be used for screening contaminants.
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References
Barrick A, Châtel A, Bruneau M, Mouneyrac C (2017) The role of high-throughput screening in ecotoxicology and engineered nanomaterials. Environ Toxicol Chem 36:1704–1714. https://doi.org/10.1002/etc.3811
Bouki E, Dimitriadis VK, Kaloyianni M, Dailianis S (2013) Antioxidant and pro-oxidant challenge of tannic acid in mussel hemocytes exposed to cadmium. Mar Environ Res 85:13–20. https://doi.org/10.1016/j.marenvres.2012.12.005
Buffet PE, Zalouk-Vergnoux A, Châtel A, Berthet B, Métais I, Perrein-Ettajani H, Poirier L, Luna-Acosta A, Thomas-Guyon H, Risso-de Faverney C, Guibbolini M, Gilliland D, Valsami-Jones E, Mouneyrac C (2014) A marine mesocosm study on the environmental fate of silver nanoparticles and toxicity effects on two endobenthic species: the ragworm Hediste diversicolor and the bivalve mollusc Scrobicularia plana. Sci Total Environ 470–471:1151–1159. https://doi.org/10.1016/j.scitotenv.2013.10.114
Canesi L, Ciacci C, Lorusso LC, Betti M, Gallo G, Pojana G, Marcomini A (2007) Effects of Triclosan on Mytilus galloprovincialis hemocyte function and digestive gland enzyme activities: possible modes of action on non target organisms. Comp Biochem Physiol C Toxicol Pharmacol 145:464–472. https://doi.org/10.1016/j.cbpc.2007.02.002
Canesi L, Ciacci C, Vallotto D, Gallo G, Marcomini A, Pojana G (2010) In vitro effects of suspensions of selected nanoparticles (C60 fullerene, TiO2, SiO2) on Mytilus hemocytes. Aquat Toxicol 96:151–158. https://doi.org/10.1016/j.aquatox.2009.10.017
Canesi L, Ciacci C, Fabbri R, Marcomini A, Pojana G, Gallo G (2012) Bivalve molluscs as a unique target group for nanoparticle toxicity. Mar Environ Res 76:16–21. https://doi.org/10.1016/j.marenvres.2011.06.005
Cao A, Mercado L, Ramos-Martinez JI, Barcia R (2003) Primary cultures of hemocytes from Mytilus galloprovincialis Lmk.: expression of IL-2Rα subunit. Aquaculture 216:1–8. https://doi.org/10.1016/S0044-8486(02)00140-0
Carballal M, López M, Azevedo C, Villalba A (1997) Hemolymph cell types of the mussel Mytilus galloprovincialis. Dis Aquat Organ 29:127–135. https://doi.org/10.3354/dao029127
Caza F, Betoulle S, Auffret M, Brousseau P, Fournier M, St-Pierre Y (2015) Comparative analysis of hemocyte properties from Mytilus edulis desolationis and Aulacomya ater in the Kerguelen Islands. Mar Environ Res 110:174–182. https://doi.org/10.1016/j.marenvres.2015.09.003
Chatziargyriou V, Dailianis S (2010) The role of selenium-dependent glutathione peroxidase (Se-GPx) against oxidative and genotoxic effects of mercury in haemocytes of mussel Mytilus galloprovincialis (Lmk.). Toxicol Vitr 24:1363–1372. https://doi.org/10.1016/j.tiv.2010.04.008
Chen J-H (1992) The cell activation model of hemocyte aggregation and adhesion in the California mussel, Mytilus Californianus. Oregon State University, College Town
Chen J-H, Bayne CJ (1995) Bivalve mollusc hemocyte behaviors: characterization of hemocyte aggregation and adhesion and their inhibition in the California mussel (Mytilus californianus). Biol Bull 188:255–266. https://doi.org/10.2307/1542303
Chevé J, Bernard G, Passelergue S, Prigent J-L (2014) Suivi bactériologique des gisements naturels de coquillages de l’Ille-et-Vilaine et des Côtes- d’Armor fréquentés en pêche à pied 1–99
Coles JA, Farley SR, Pipe RK (1995) Alteration of the immune response of the common marine mussel Mytilus edulis resulting from exposure to cadmium. Dis Aquat Org 22:59–65. https://doi.org/10.3354/dao022059
Comps M, Tigé G (1999) Procaryotic infections in the mussel Mytilus galloprovinciallis and in its parasite the turbellarian Urastoma cyprinae. Dis Aquat Org 38:211–217. https://doi.org/10.3354/dao038211
Conrad P, Atwill E, Garner I, Miller M, Leutenegger C, Arkush K, Jesuup D (2005) Cryptosporidium in bivalves as indicators of fecal pollution in the California coastal ecosystem. UC Berkeley Tech. Complet. Reports
Daugavet MA, Blinova MI (2015) Culture of mussel (Mytiuls edulis L.) mantle cells. Cell Tissue Biol 9:233–243. https://doi.org/10.1134/S1990519X15030037
Domart-Coulon I, Auzoux-Bordenave S, Doumenc D, Khalanski M (2000) Cytotoxicity assessment of antibiofouling compounds and by-products in marine bivalve cell cultures. Toxicol Vitr 14:245–251. https://doi.org/10.1016/S0887-2333(00)00011-4
Droguet M (2006) Etude des caracteristiques fonctionnelles des cardiomyocete d’huitre en culture. Universite de Bretagne Occidentale
Droguet M, Devauchelle N, Pennec J-P, Quinn B, Dorange G (2012) Cultured heart cells from oyster: an experimental approach for evaluation of the toxicity of the marine pollutant tributyltin. Aquat Living Resour 25:185–194. https://doi.org/10.1051/alr/2012017
Faucet J, Maurice M, Gagnaire B, Renault T, Burgeot T (2004) Isolation and primary culture of gill and digestive gland cells from the common mussel Mytilus edulis. Methods Cell Sci 25:177–184. https://doi.org/10.1007/s11022-004-8227-4
Fenwick N, Griffin G, Gauthier C (2009) The welfare of animals used in science: how the “Three Rs” ethic guides improvements. Can Vet J 50:523–530
Freshney IR (2016) Culture of animal cells: a manual of basic technique and specialized applications, 7th edn. Wiley, London
García-García E, Prado-Álvarez M, Novoa B, Figueras A, Rosales C (2008) Immune responses of mussel hemocyte subpopulations are differentially regulated by enzymes of the PI 3-K, PKC, and ERK kinase families. Dev Comp Immunol 32:637–653. https://doi.org/10.1016/j.dci.2007.10.004
Gómez-Mendikute A, Cajaraville MP (2003) Comparative effects of cadmium, copper, paraquat and benzo[a]pyrene on the actin cytoskeleton and production of reactive oxygen species (ROS) in mussel haemocytes. Toxicol Vitr 17:539–546. https://doi.org/10.1016/S0887-2333(03)00093-6
Gomez-Mendikute A, Etxeberria A, Olabarrieta I, Cajaraville MP (2002) Oxygen radicals production and actin filament disruption in bivalve haemocytes treated with benzo(a)pyrene. Mar Environ Res 54:431–436. https://doi.org/10.1016/S0141-1136(02)00177-0
Gustafson LL, Stoskopf MK, Bogan AE, Showers W, Kwak TJ, Hanlon S, Levine JF (2005) Evaluation of a nonlethal technique for hemolymph collection in Elliptio complanata, a freshwater bivalve (Mollusca: Unionidae). Dis Aquat Org 65:159–165. https://doi.org/10.3354/dao065159
Hanana H, Talarmin H, Pennec JP, Droguet M, Gobin E, Marcorelle P, Dorange G (2011) Establishment of functional primary cultures of heart cells from the clam Ruditapes decussatus. Cytotechnology 63:295–305. https://doi.org/10.1007/s10616-011-9347-8
Höher N, Turja R, Köhler A, Lehtonen KK, Broeg K (2015) Immunological responses in the mussel Mytilus trossulus transplanted at the coastline of the northern Baltic Sea. Mar Environ Res 112:113–121. https://doi.org/10.1016/j.marenvres.2015.10.003
Jimeno-Romero A, Bilbao E, Izagirre U, Cajaraville MP, Marigómez I, Soto M (2017) Digestive cell lysosomes as main targets for Ag accumulation and toxicity in marine mussels, Mytilus galloprovincialis, exposed to maltose-stabilised Ag nanoparticles of different sizes. Nanotoxicology 11:168–183. https://doi.org/10.1080/17435390.2017.1279358
Judson R, Kavlock R, Martin M, Reif D, Houck K (2013) Perspectives on validation of high-throughput assays supporting 21st century toxicity testing. ALTEX 30:51–66
Katsumiti A, Berhanu D, Howard KT, Arostegui I, Oron M, Reip P, Valsami-Jones E, Cajaraville MP (2014a) Cytotoxicity of TiO2 nanoparticles to mussel hemocytes and gill cells in vitro: influence of synthesis method, crystalline structure, size and additive. Nanotoxicology 9:543–553. https://doi.org/10.3109/17435390.2014.952362
Katsumiti A, Gilliland D, Arostegui I, Cajaraville MP (2014b) Cytotoxicity and cellular mechanisms involved in the toxicity of CdS quantum dots in hemocytes and gill cells of the mussel Mytilus galloprovincialis. Aquat Toxicol 153:39–52. https://doi.org/10.1016/j.aquatox.2014.02.003
Katsumiti A, Arostegui I, Oron M, Gilliland D, Valsami-Jones E, Cajaraville MP (2016) Cytotoxicity of Au, ZnO and SiO2 NPs using in vitro assays with mussel hemocytes and gill cells: relevance of size, shape and additives. Nanotoxicology 10:185–193. https://doi.org/10.3109/17435390.2015.1039092
Le Marrec-Croq F, Glaise D, Guguen-Guillouzo C, Chesne C, Guillouzo A, Boulo V, Dorange G (1999) Primary cultures of heart cells from the scallop Pecten maximus (mollusca-bivalvia). In Vitro Cell Dev Biol Anim 35:289–295
Le Pennec G, Le Pennec M (2001) Acinar primary cell culture from the digestive gland of Pecten maximus (L.): an original model for ecotoxicological purposes. J Exp Mar Bio Ecol 259:171–187. https://doi.org/10.1016/S0022-0981(01)00232-5
Leverett D, Thain J (2013) ICES techniques in marine environmental sciences: oyster embryo-larval bioassay (revised). ICES Tech Mar Environ Sci 54. https://doi.org/10.13140/RG.2.1.3484.6165
Martínez-Gómez C, Bignell J, Lowe D (2015) Lysosomal membrane stability in mussels. ICES Tech Mar Environ Sci 56:41
Moore MN, Lowe DM (1977) The cytology and cytochemistry of the hemocytes of Mytilus edulis and their responses to experimentally injected carbon particles. J Invertebr Pathol 29:18–30. https://doi.org/10.1016/0022-2011(77)90167-7
Mothersill C, Austin B (2000) Aquatic invertebrate cell culture. Springer, Berlin
National Institute of Environmental and Health Sciences (2001) Guidance document on using In vitro data to estimate in vivo starting doses for acute toxicity. Natl Toxicol Progr Interag Cent Eval Altern Toxicol Methods 1–102
OECD Environment Directorate (2017) Alternative testing strategies in risk assessment of manufactured nanomaterials: current state of knowledge and research needs to advance their use. OECD Environment, Health and Safety Publications Series on Safety of Manufactured Nanomaterials. 80:JT03408320
Quinn B, Costello MJ, Dorange G, Wilson JG, Mothersill C (2009) Development of an in vitro culture method for cells and tissues from the zebra mussel (Dreissena polymorpha). Cytotechnology 59:121–134. https://doi.org/10.1007/s10616-009-9202-3
Rampersad SN (2012) Multiple applications of alamar blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors (Switzerland) 12:12347–12360. https://doi.org/10.3390/s120912347
Renwrantz L, Siegmund E, Woldmann M (2013) Variations in hemocyte counts in the mussel, Mytilus edulis: similar reaction patterns occur in disappearance and return of molluscan hemocytes and vertebrate leukocytes. Comp. Biochem Physiol A Mol Integr Physiol 164:629–637. https://doi.org/10.1016/j.cbpa.2013.01.021
Rioult D, Lebel JM, Le Foll F (2013) Cell tracking and velocimetric parameters analysis as an approach to assess activity of mussel (Mytilus edulis) hemocytes in vitro. Cytotechnology 65:749–758. https://doi.org/10.1007/s10616-013-9558-2
St-Jean SD, Courtenay SC, Parker RW (2003) Immunomodulation in Blue Mussels (Mytilus edulis) exposed to a pulp and paper mill effluent in eastern Canada. Water Qual Res 38:647–666
Stone V, Pozzi-Mucelli S, Tran L, Aschberger K, Sabella S, Vogel U, Poland C, Balharry D, Fernandes T, Gottardo S, Hankin S, Hartl MG, Hartmann N, Hristozov D, Hund-Rinke K, Johnston H, Marcomini A, Panzer O, Roncato D, Saber AT, Wallin H, Scott-Fordsmand JJ (2014) ITS-NANO—prioritising nanosafety research to develop a stakeholder driven intelligent testing strategy. Part Fibre Toxicol 11:9. https://doi.org/10.1186/1743-8977-11-9
Tanguy M, McKenna P, Gauthier-Clerc S, Pellerin J, Danger JM, Siah A (2013) Sequence analysis of a normalized cDNA library of Mytilus edulis hemocytes exposed to Vibrio splendidus LGP32 strain. Results Immunol 3:40–50. https://doi.org/10.1016/j.rinim.2013.04.001
Villalba A, Mourelle SG, Carballal MJ, López C (1997) Symbionts and diseases of farmed mussels Mytilus galloprovincialis throughout the culture process in the Rias of Galicia (NW Spain). Dis Aquat Org 31:127–139. https://doi.org/10.3354/dao031127
Yoshino TP, Bickham U, Bayne CJ (2013) Molluscan cells in culture: primary cell cultures and cell lines. Can J Zool 91:391–404. https://doi.org/10.1139/cjz-2012-0258
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Barrick, A., Guillet, C., Mouneyrac, C. et al. Investigating the establishment of primary cultures of hemocytes from Mytilus edulis. Cytotechnology 70, 1205–1220 (2018). https://doi.org/10.1007/s10616-018-0212-x
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DOI: https://doi.org/10.1007/s10616-018-0212-x