Isolation of cells from a Marine Source Responsible for Hemocyanin Biosynthesis

Abstract

Molluscan rhogocyte cells are known to synthesise the largest respiratory proteins in nature known as hemocyanin. However, attempt to investigate the cells in vitro to understand the hemocyanin biosynthesis is difficult due to the lack of current knowledge on establishing a stable cell culture. Therefore, the aims of this study were to isolate rhogocyte cells from Haliotis laevigata and assessed their characteristics, distribution, capability to grow and synthesise hemocyanin in vitro. Using flow cytometry analysis and simultaneous staining of immunofluorescence and in situ hybridisation strategy, two distinct populations of rhogocyte cells synthesizing type 1 hemocyanin was determined in the mantle tissue. Subsequently, a primary culture of heterogeneous cells was established with different parameters involving basal media, primary growth supplements, secondary growth supplements, growth temperature and seeding density. Cells cultured in MEM result in the highest cell yield compared to other basal media. It is suggested that the presence of vitamin B6 in aldehyde form (pyridoxal) instead of alcohol (pyridoxine) is responsible for promoting the cellular activity. Furthermore, addition of lipimax at 17°C resulted in higher cell-fold increase compared to FBS, knockout serum and hemolymph. Addition of secondary supplements such as amino acids cocktail, lipid concentrate, insulin-transferrin-selenium and vitamin concentrate, however, had no significant impact on the cell growth. On the other hand, evaluation of hemocyanin content using ELISA revealed significant increase of hemocyanin in the media when cells were cultured with lipimax, FBS and knockout serum. However, the hemocyanin content was at the highest concentration only after an hour of culture before decreasing significantly and stabilised around 0.14-0.16 µg/ml in the media. These results suggest that hemocyanin biosynthesis may have an inverse correlation with the cell’s growth.