Summary
Conditions were developed to cryopreserve cells from pronase-dissociated atria and ventricles of eastern oysters (Crassostrea virginica). The effect of three concentrations (5, 10, 15%) of the cryoprotectants (dimethyl sulfoxide, glycerol, and propylene glycol), three thawing temperatures (25, 45, 75°C), and three cooling rates (slow, medium, fast) were compared. Cells were frozen at −80°C and plunged in liquid nitrogen. Thawed cells were seeded in 96-well plates and primary cultures were evaluated after 3 d by measuring the metabolic activity using a tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-( 3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, and by comparing the relative spreading of cells between treatments. The best conditions for freezing and thawing of cells for each cryoprotectant were selected and a final study was performed to compare cryoprotectants. For this final study, we measured the number of cells and their viability 3 d after thawing, in addition to determining cell metabolic activity and cell spreading. Primary cultures of cells frozen without cryoprotectant and of nonfrozen cells were used as controls in all studies. Atrial cells were best cryopreserved with glycerol at a concentration of 10%, a medium cooling rate, and thawing at 45°C. After thawing, atrial cells showed 53±5% of the metabolic activity, 84±5% of the number, and 92±2% of the viability of nonfrozen cells. For ventricular cells, 10% glycerol, a medium cooling rate, and thawing at 25°C yielded the best results. The thawed ventricular cells showed 83±5% of the metabolic activity, 91±5% of the number, and 96±2% of the viability of nonfrozen cells.
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References
Berthelin, C.; Kellner, K.; Mathieu, M. Histological characterization and glucose incorporation into glycogen of the Pacific oysterCrassostrea gigas storage cells. Mar. Biotechnol. 2:136–145; 2000.
Birmelin, C.; Pipe, R. K.; Goldfarb, P. S., et al. Primary cell-culture of the digestive gland of the marine musselMytilus edulis: a time-course study of antioxidant- and biotransformation-enzyme activity and ultrastructural changes. Mar. Biol. 135:65–75; 1999.
Boulo, V.; Cadoret, J. P.; Le Marrec, F., et al. Transient expression of luciferase reporter genes after lipofection in oyster (Crassostrea gigas) primary cultures. Mol. Mar. Biol. Biotechnol. 5:167–174; 1996.
Brewster, F.; Nicholson, B. L.In vitro maintenance of amoebocytes from the American oysterCrassostrea virginica). J. Fish. Res. Bd. Can. 36:461–467; 1979.
Buchanan, J. T.; La Peyre, J. F.; Cooper, R. K., et al. Improved attachment and spreading in primary cell cultures of the eastern oyster,Crassostrea virginica. In Vitro Cell. Dey. Biol. 35A:593–598; 1999.
Canesi, L.; Ciacci, C.; Betti, M., et al. Growth factors stimulate the activity of key glycolytic enzymes in isolated digestive gland cells from mussels (Mytilus galloprovincialis Lam.) through tyrosine kinase signal transduction. Gen. Comp. Endocrinol. 116:241–248; 1999.
Chao, N. H.; Lin, T. T.; Chen, Y. J., et al. Cryopreservation of late embryos and early larvae in the oyster and hard clam. Aquaculture 155:31–44; 1997.
Chen, S.-N.; Wen, C.-M. Establishment of cell lines derived from oyster,Crassostrea gigas Thunberg and hard clam,Meretrix lusoria Roding. Methods Cell Sci. 21(4):183–192; 1999.
Freshney, R. I. Culture of animal cells: a manual of basic technique. 3rd ed. New York: Wiley-Liss; 1994.
Gabbott, P. A. Developmental and seasonal metabolic activities in marine molluscs. In: Hochachka, P. W., ed. The Mollusca, Vol. 2F. New York: Academic Press 1983:165–217.
Gwo, J. C. Cryopreservation of oyster (Crassostrea gigas) embryos. Theriogenology 43:1163–1174; 1995.
Gwo, J. C. Cryopreservation of aquatic invertebrate semen: a review. Aquacult. Res. 31:259–271; 2000.
Hetrick, F. M.: Stephens, E.; Lomax, N., et al. Attempts to develop a marine molluscan cell line. College Park, University of Maryland. Sea Grant College Program Technical Report UM-SG-TS-81-06; 1981.
Horita, C.; Mega, H.; Kurokura, H. Grafting of cryopreserved mantle tissues onto cultured oyster pearl oyster. Cryo-Lett. 20:311–314, 1999.
La Peyre, J. F.; Faisal, F. M.; Burreson, E. M.In vitro propagation of the protozoanPerkinsus marinus a pathogen of the Eastern oysterCrassostrea virginica. J. Eukaryot. Microbiol. 40:304–310; 1993.
Le Marrec-Croq, F.; Fritayre, P.; Chesn, A., et al. Cryopreservation ofPecten maximus heart cells. Cryobiology 37:200–206; 1998.
Leung, C. K. P. Principles of biological cryopreservation. In: Jamieson, B. G. M., ed. Fish evolution and systematics: evidence from spermatozoa. New York: Cambridge University Press; 1991;231–269.
Li, M. F.; Stewart, J. E.; Drinnan, R. E.In vitro cultivation of cells of the oyster,Crassostrea virginica. J. Fish. Res. Bd. Can. 23:595–599; 1966.
Lin, T. T.; Chao, N. H.; Tung, H. T. Factors affecting survival of cryopreserved oyster (Crassostrea gigas) embryos. Cryobiology 39:192–196; 1999.
Loomis, S. H.; Carpenter, J. F.; Crowe, J. H. Identification of strombine and taurine as cryoprotectants in the intertidal bivalveMytilus edulis. Biochim. Biophys. Acta 943:113–118; 1988.
Lovelock, J. E.; Bishop, M. W. H. Prevention of cooling damage to living cells by dimethyl sulfoxide. Nature 183:1394–1395; 1959.
Naidenko, T. Cryopreservation ofCrassostrea gigas oocytes, embryo and larvae using antioxidant echinochrome and an antifreeze protein AFP1. Cryo-Lett. 18:375–382; 1997.
Odintsova, N.; Tsal, L. Cryopreservation of primary cell cultures of bivalvia. Cryo-Lett. 16:13–20; 1995.
Paniagua-Chavez, C. G.; Buchanan, J. T.; Supan, J. E., et al. Settlement and growth of eastern oysters produced from cryopreserved larvae. Cryo-Lett. 19:283–292; 1998.
Pazos, A. J.; Mathieu, M. Effects of five natural gonadotropin-releasing hormones on cell suspensions of marine bivalve gonad: stimulation of gonial DNA synthesis. Gen. Comp. Endocrinol. 113:112–120; 1999.
Pomponi, S. A.; Willoughby, R.; Kaighn, M. E., et al. Development of techniques forin vitro production of bioactive natural products from marine sponges. In: Maramorosh, K., Mitsuhashi, J., ed. Novel directions and biotechnology applications. New Hampshire: Science Publishers; 1997:231–137.
Rinkevich, B. Cell cultures from marine invertebrates: obstacles, new approaches and recent improvements. J. Biochenol. 70:133–153; 1999.
Shumway, S. E. Natural enyironmental factors. In: Kennedy, V.; Newell, R. I. E.; Eble, A. F., ed. The eastern oyster,Crassostrea virginica. College Park, MD: Maryland Sea Grant College, University of Maryland System; 1996:467–513.
Stephens, E. B.; Hetrick, F. M. Decontamination of the American oyster tissues for cell and organ culture. TCA Manual 5:1029–1031; 1979.
Thompson, R. J.; Newell, R. I. E.; Kennedy, V. S., et al. Reproductive processes and early development. In: Kennedy, V.; Newell, R. I. E.; Eble, A. F., ed. The eastern oyster,Crassostrea virginica. College Park, MD: Maryland Sea Grant College, University of Maryland System; 1996:335–370.
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Cheng, T.C., La Peyre, J.F., Buchanan, J.T. et al. Cryopreservation of heart cells from the eastern oyster. In Vitro Cell.Dev.Biol.-Animal 37, 237–243 (2001). https://doi.org/10.1007/BF02577536
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DOI: https://doi.org/10.1007/BF02577536