Development of an in vitro culture method for cells and tissues from the zebra mussel (Dreissena polymorpha)

Abstract

Despite the successful transfer of mammalian in vitro techniques for use with fish and other vertebrates, little progress has been made in the area of invertebrate tissue culture. This paper describes the development of an in vitro technique for the culture of both cells in suspension and tissue explants from the gill, digestive gland and mantle of the zebra mussel (Dreissena polymorpha) and their successful maintenance in culture for up to 14 days. Cell suspensions from the gills and digestive gland were the most successful technique developed with viability [80% maintained for up to 8 days in culture, suitable for use in short term toxicity tests. Tissue explants from the mantle were also maintained in culture for up to 14 days. This paper describes the challenges involved in the development of a novel in vitro culture technique for aquatic invertebrates.

Does zebra mussel (Dreissena polymorpha) represent the freshwater counterpart of Mytilus in ecotoxicological studies? A critical review

“…Considering all these reasons, cultures of different vertebrate cell lines have been standardized and widely applied in ecotoxicology, but only recently cells from different organs of marine bivalves, including gills (G omez- Mendikute et al, 2005), mantle (Koyama and Aizawa, 2000;Barik et al, 2004;Cornet, 2006), digestive gland (Le Le Penec, 2001, 2003;Chelomin et al, 2005) and hearth (DomartCoulon et al, 2000) have been cultured. To fill this gap for freshwater environments, Quinn et al (2009) developed an in vitro technique for culturing cells in suspension and tissue explants from the gill, digestive gland and mantle of the zebra mussel, showing their successful maintenance in culture for up to 14 days. The final goal of this technique development was for its potential use in toxicity tests to assess the mechanistic effect of different environmental pollutants on isolated cells and tissues (Quinn et al, 2009).…”

Establishment of functional primary cultures of heart cells from the clam Ruditapes decussatus

“…Numerous attempts in culturing cells have been performed from different marine or freshwater bivalves (Rinkevich 2005) such as clams (Cecil 1969;Wen et al 1993b;Chen and Wen 1999), mussels (Chardonnet and Pérès 1963;Quinn et al 2009), scallops (Le Marrec-Croq et al 1998Fritayre 2004;Talarmin et al unpublished) and oysters (e.g. Le Deuff et al 1994;Renault et al 1995;Buchanan et al 1999;Chen and Wen 1999;DomartCoulon et al 2000;Pennec et al 2002Pennec et al , 2004Droguet 2006;Talarmin et al 2008).…”

Isolation and fractionation of gill cells from freshwater (Lasmigona costata) and seawater (Mesodesma mactroides) bivalves for use in toxicological studies with copper

“…In contrast, our data indicate that the enzymatic method was an excellent option to obtain isolated cells from gills of L. costata. Pronase, the enzyme used in the present study, has been described as the most satisfactory in cell dissociation in the freshwater mussel Dreissena polymorpha (Quinn et al 2009). However, mechanical shaking was included and the time of tissue incubation with the enzyme was improved (reduced from 16 h to 20 min) in the protocol applied for L. costata in the present study, thus resulting in a satisfactory number of isolated cells (*10 7 cells) at a high percentage of viability in a shorter time.…”

“…However, the higher percentage of cells rich in mitochondria found in the present study suggests that there could have been enrichment associated with the cellular dissociation technique employed. Although it is regarded as the best method for cell isolation in the freshwater bivalve Dreissena polymorpha (Quinn et al 2009), enzymatic digestion is considered to be an ''aggressive'' method to perform cell isolation. Therefore, we suspect that a large proportion of the debris and dead cells found after applying the enzymatic method in L. costata, i.e., tissue digestion with pronase, may have been derived from cells showing low mitochondrial density and low Na ?…”

“…However, the maintenance of small pieces of gill tissue in freshwater phosphate buffer solution (PBS) for 40 min caused massive cell death (data not shown). Therefore, we subsequently employed enzymatic dissociation as described by Quinn et al (2009) with modifications. Briefly, gill tissue from two mussels were dissected, pooled, and held in calciumfree phosphate buffer solution (freshwater PBS; composition in mM: 9 NaCl, 5 NaHCO 3 , 0.5 KCl, 5 Na 2 PO 4 ; pH 7.6; 20°C) for 10 min to remove the excess mucus.…”