Abstract
Development of protocols and media for culturing immune cells from marine invertebrates has not kept pace with advancements in mammalian immune cell culture, the latter having been driven by the need to understand the causes of and develop therapies for human and animal diseases. However, expansion of the aquaculture industry and the diseases that threaten these systems creates the need to develop cell and tissue culture methods for marine invertebrates. Such methods will enable us to better understand the causes of disease outbreaks and to develop means to avoid and remedy epidemics. We report a method for the short-term culture of phagocytes from the purple sea urchin, Strongylocentrotus purpuratus, by modifying an approach previously used to culture cells from another sea urchin species. The viability of cultured phagocytes from the purple sea urchin decreases from 91.6% to 57% over six days and phagocyte morphology changes from single cells to aggregates leading to the formation of syncytia-like structures. This process is accelerated in the presence of lipopolysaccharide suggesting that phagocytes are capable of detecting this molecular pattern in culture conditions. Sea urchin immune response proteins, called Sp185/333, are expressed on the surface of a subset of phagocytes and have been associated with syncytia-like structures. We evaluated their expression in cultured phagocytes to determine their possible role in cell aggregation and in the formation of syncytia-like structures. Between 0 and 3 hr, syncytia-like structures were observed in cultures when only ∼10% of the cells were positive for Sp185/333 proteins. At 24 hr, ∼90% of the nuclei were Sp185/333-positive when all of the phagocytes had aggregated into syncytia-like structures. Consequently, we conclude that the Sp185/333 proteins do not have a major role in initiating the aggregation of cultured phagocytes, however the Sp185/333 proteins are associated with the clustered nuclei within the syncytia-like structures.
Highlights
The advancement of methods for culturing mammalian immune cells has proceeded ahead of progress for developing cell culture methods for lower vertebrates and invertebrates
Echinoid Coelomocyte Culture Medium Seven different culture media were tested on phagocytes to determine which one was optimal for subsequent studies of cells in short-term primary cultures (Table 1)
echinoid coelomocyte culture medium (ECCM) included the original LDF recipe diluted into artificial coelomic fluid (aCF) with modifications, with the addition of 10% cell-free coelomic fluid and the omission of growth factors (Table 1)
Summary
The advancement of methods for culturing mammalian immune cells has proceeded ahead of progress for developing cell culture methods for lower vertebrates and invertebrates. Cell and tissue culture systems provide tools to study basic biological functions of marine organisms including the evaluation of responses to toxins and pathogens. Several marine invertebrates have been subjects of cell culture studies, including species of shrimp, crabs, crayfish, molluscs, ascidians, sea stars, sea cucumbers and sea urchins (reviewed in [2]). The subject of this study, the California purple sea urchin, Strongylocentrotus purpuratus, is important to aquaculture (mainly for harvesting ovaries [Japanese uni] for human consumption), but these animals have been used in studies of developmental biology for over 100 years (reviewed in [3]). Because sea urchins and other classes of echinoderms are close relatives to the chordates, these species provide clues to important evolutionary processes within the deuterostome lineage of animals that includes the vertebrates [4]
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