Influence of cryoprotective agent and cooling rate on frozen and thawed hemocytes from the Mollusk Haliotis tuberculata.

Cultures of circulating cells from abalone (Haliotis tuberculata) may be used in fundamental research or in biotechnology. This paper describes attempts to develop a cryoconservation method for these hemocytes in order to constitute a standardized cell stock. Among a panel of five distinct cryoprotective solutions, 10% v/v glycerol ('G solution') was the most effective and better post-thaw recovery was achieved after cooling at 1 degrees C/min than after more rapid cooling (3 degrees C or 9 degrees C/min). In 2-day-old cultures, cell viability, assessed by DNA or protein content, was 83 and 78%, respectively, and metabolic activity, measured by the MTT reduction assay, reached 96%. Viability rates were only slightly reduced after 6 days of culture, suggesting a low proportion of damaged cells among the surviving hemocytes. This study identified a cryoprotective solution and a freezing protocol that allow thawed hemocytes to recover a large part of their viability.