Isolation of coelomocyte from sea urchin Echinometra mathaei: optimization of culture condition

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Abstract
Rapid growth of the aquaculture industry is hampered by infectious diseases in marine invertebrates, causing economic losses. Marine invertebrate cell cultures offer tools to evaluate biological properties and cellular responses in different conditions. Long-term culture aims to isolate tissue-specific cells and identify bioactive compounds from stem cells. Echinometra mathaei, known as Persian Gulf sea urchin, has lots of benefits in various fields including aquaculture, embryology, and evolutionary biology. However, its cell culture faces challenges due to poorly characterized microenvironmental and specific cultivation requirements. This study aims to establish and optimize a long-term cell culture for coelomocyte derived from E. mathaei, focusing on the characterization of microenvironment conditions to overcome the limitations of current marine invertebrate cell culture. After the collection of E. mathaei from Lark Island, Persian Gulf, Iran, and their acclimatization in artificial seawater, coelomocytes were isolated from different sources including the coelomic fluid, the coelomic epithelium, and the axial organ. Various cell dissociation methods, culture media, growth supplements, culture dishes, and physical conditions were tested to determine optimal conditions for coelomocyte in vitro culture. Moreover, coelomocytes were differentiated to pigment-producing cells, and naphthoquinone pigments were extracted and identified using spectrophotometry. Light microscopy identified several coelomocyte types, including petaloid, filopodial, vibratile cells, and spherulocytes. The HCCM medium supplemented with coelomic fluid proved most effective for cell growth and viability. Moreover, coelomic fluid is the best culture media for differentiation of coelomocyte into the cell producing naphthoquinone pigments. These findings contribute to developing in vitro cell culture methods for sea urchin, providing a foundation for further research on sea urchin immunology, cell biology, and cellular responses to pathogens and other biological stress.

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