Cryopreservation ofPecten maximusHeart Cells

Marrec-Croq, Françoise Le; Fritayre, P; Chesné, Christophe; Guillouzo, André; Dorange, Germaine

doi:10.1006/cryo.1998.2113

Cited by 19 publications

(16 citation statements)

References 10 publications

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“…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mussels (Chardonnet and Pérès 1963;Quinn et al 2009), scallops (Le Marrec-Croq et al 1998Fritayre 2004;Talarmin et al unpublished) and oysters (e.g. Le Deuff et al 1994;Renault et al 1995;Buchanan et al 1999;Chen and Wen 1999;DomartCoulon et al 2000;Pennec et al 2002Pennec et al ,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).…”

Section: Introductionmentioning

confidence: 99%

“…from oysters and scallops can be now routinely maintained functional in culture up to 1 month (Domart-Coulon et al 1994;Le MarrecCroq et al 1997, 1998Pennec et al 2002Pennec et al , 2004Fritayre 2004;Droguet 2006;Talarmin et al 2008). Primary cultures can be obtained from freshly isolated cardiomyocytes and from cryopreserved cells (Le Marrec-Croq et al 1998;Cheng et al 2001).…”

Section: Introductionmentioning

confidence: 99%

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

Hanana¹,

Talarmin²,

Pennec³

et al. 2011

Cytotechnology

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Heart cells from the clam Ruditapes decussatus were routinely cultured with a high level of reproducibility in sea water based medium. Three cell types attached to the plastic after 2 days and could be maintained in vitro for at least 1 month: epithelial-like cells, round cells and fibroblastic cells. Fibroblastic cells were identified as functional cardiomyocytes due to their spontaneous beating, their ultrastructural characteristics and their reactivity with antibodies against sarcomeric a-actinin, sarcomeric tropomyosin, myosin and troponin T-C. Patch clamp measurements allowed the identification of ionic currents characteristic of cardiomyocytes: a delayed potassium current (I K slow ) strongly suppressed (95%) by tetraethylammonium (1 mM), a fast inactivating potassium current (I K fast ) inhibited (50%) by 4 amino-pyridine at 1 mM and, at a lower level (34%) by TEA, a calcium dependent potassium current (I KCa ) activated by strong depolarization. Three inward voltage activated currents were also characterized in some cardiomyocytes: L-type calcium current (I Ca ) inhibited by verapamil at 5 9 10 -4 M, T-type Ca 2? current, rapidly activated and inactivated, and sodium current (I Na ) observed in only a few cells after strong hyperpolarization. These two currents did not seem to be physiologically essential in the initiation of the beatings of cardiomyocytes. Potassium currents were partially inhibited by tributyltin (TBT) (1 lM) but not by okadaic acid (two marine pollutants). DNA synthesis was also demonstrated in few cultured cells using BrdU (bromo-2 0 -deoxyuridine). Observed effects of okadaic acid and TBT demonstrated that cultured heart cells from clam Ruditapes decussatus can be used as an experimental model in marine toxicology.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Hanana¹,

Talarmin²,

Pennec³

et al. 2011

Cytotechnology

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show abstract

“…Primary cultures of oyster cardiac cells were successfully developed in our laboratory and are now routinely obtained in a similar way to our successful cultures of scallop and clam heart cells (Le Marrec-Croq et al 1998Pennec et al 2002Pennec et al , 2004Droguet 2006;Hanana 2011;Hanana et al 2011). An interesting point about these different species is that, in the natural environment, they are representative of different ecosystems where TBT pollution may occur.…”

Section: Discussionmentioning

confidence: 99%

“…However, functional primary cultures have been frequently established using heart cells of bivalves (e.g., Le Marrec-Croq et al 1999;Pennec et al 2002Pennec et al , 2004Talarmin et al 2008;Hanana 2011), especially the oyster Crassostrea gigas. Such cell cultures can be obtained from freshly isolated or cryopreserved cells (Pennec et al 2004) using a freeze/thaw protocol defined in the scallop Pecten maximus by Le Marrec-Croq et al (1998) that was optimised in C. gigas by Droguet (2006), thus providing a constant standardized source of cells for applied research.…”

Section: Introductionmentioning

confidence: 99%

Cultured heart cells from oyster : an experimental approach for evaluation of the toxicity of the marine pollutant tributyltin

Droguet

Devauchelle

Pennec

et al. 2012

Aquat. Living Resour.

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-European Community regulations on chemicals promote alternative methods to test substances presenting potential risks for the environment. In the present work, cultured atrial cells isolated from oyster (Crassostrea gigas) were used as an experimental model to investigate the toxicity of tributyltin (TBT) after short-time exposure at concentrations representative of those that can be measured in seawater, marine sediments and/or bivalves bioaccumulating this pollutant. In vitro and in vivo assays produce values of the same order of magnitude for both animal/cell survival and heart/cardiomyocyte beating rate. The survival rate of whole animals decreased from 10 −6 M TBT after 3 days. For cultured cells, the viability, evaluated using 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay, significantly decreased after two days of treatment with 10 −6 M TBT, and after six days with 10 −10 M TBT. The percentage of apoptotic cells, quantified by flow cytometry and YO-PRO r -1 iodide, a nucleic acid stain that only permeates cells that are beginning to undergo apoptosis, increased significantly in these cases. Moreover, intracellular concentration of Ca ++ had increased after 10 min of exposition to 10 −6 M, and could be associated with apoptotic processes. As patch clamp experiments showed that Ca ++ conductance was decreased, intracellular calcium increase could mainly be due to a release from internal stores. The decreases in beating rhythm could be explained by the decrease in adenosine triphosphate (ATP) production revealed by 31 P nuclear magnetic resonance (NMR) spectroscopy and confirmed by the increase of the K ATP channel conductance. The related hyperpolarization and the disturbances of the energetic metabolism were clearly related to the loss of the atrial cell contractility and viability.

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“…This lack of progress is partially due to very limited knowledge about the nutritional requirements of invertebrate cells, the use of materials and media developed for mammalian cells, as opposed invertebrate-specific and the enormous number of variables including dissection, decontamination, dissociation and culture environment (media development) involved in establishing a new in vitro technique. Despite this some success has been achieved with the reported in vitro maintenance of heart cells of the scallop Pecten maximus (Le Marrec-Croq et al 1998), soft-shell clam Mya arenaria (Kleinschuster et al 1996) and oyster Crassostrea spp (Buchanan et al 1999;Domart-Coulon et al 2000). Primary culture of gill cells from Mytilus galloprovincialis (GomezMendikute et al 2005) and mantle cells of the blue mussel M. galloprovincialis (Cornet 2006), the freshwater bivalve Lamellidens marginalis (Barik et al 2004) and the deep sea clam Calyptogena soyoae (Koyama and Aizawa 2000) were also reported.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2009

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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.

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Cryopreservation ofPecten maximusHeart Cells

Cited by 19 publications

References 10 publications

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

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

Cultured heart cells from oyster : an experimental approach for evaluation of the toxicity of the marine pollutant tributyltin

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

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