Improved attachment and spreading in primary cell cultures of the eastern oyster, Crassostrea virginica

Summary

At present, establishment of a cell line from bivalve molluscs has been unsuccessful, and in vitro work is limited to primary cell cultures. We sought to improve attachment and spreading of cells of the eastern oyster, Crassostrea virginica, to aid primary cultures and to assist development of a bivalve cell line. Our objectives were to examine the effects of substrate on ventricle cell viability, attachment, and spreading by testing of collagen I, collagen IV, fibronectin, laminin, poly-d-lysine, and two types of uncoated tissue culture plates (Falcon® and Corning®). Experiments were conducted by incubating cells with the various substrates for 24 h and 5 d. An assay with a tetrazolium compound (MTS) was used to estimate cell numbers based on metabolic activity. Although differences in MTS assay values for substrate effect on cell viability were detected at 24 h and at 5 d (P>0.0001), these were attributed to variations in metabolic activity due to different levels of attachment and spreading among treatments. Differences among treatments were detected in attachment and spreading at 24 h and 5 d (for all, P>0.0001). At 24 h, poly-d-lysine induced the highest levels of attachment and spreading; no other factor performed better than the uncoated Falcon® substrate, and collagen I performed most poorly. At 5 d, poly-d-lysine and the uncoated Corning® substrate induced significantly higher levels of attachment and spreading than did the uncoated Falcon® substrate, and collagen I performed most poorly. From these results, poly-d-lysine best promoted cell attachment and spreading. Fibronectin (at 24 h) and laminin (at 5 d) warrant further study. Along with improvements in medium composition, future work should involve screening of other attachment factors and combinations of factors, including those of bivalve origin.