Studies On The Identification, Purification And Culture In Vitro Of Archaeocytes From Marine Sponge Hymeniacicon Perleve

The lowest multi-cellular invertebrate animals– sponges (Porifera) possess an exceptional diversity of bioactive metabolites for new drug development. However the quantity of extractable sponge biomass available from the ocean cannot usually meet the demand for large scale research and clinical development of these compounds. Among various technologies proposed to solve this problem, in vitro cultivation of marine sponge cells has been considered as a promising approach. This area of research has therefore attracted great attention, although limited success has been achieved, with no single permanent proliferating cell line established.Archaeocytes are indispensable cell types, which are generally considered as the toti/multipotent'stem cells'in sponges. They have the capacity to proliferate, and differentiate into other cell types. Based on this rationale we have focused our efforts in the development of archaeocyte culture in vitro.To investigate whether the proliferating cells in primmorph cultures are indeed associated with archaeocytes, a prerequisite is to develop techniques for identifying and obtaining highly pure archaeocytes.Firstly we investigated the structural characteristics of the main cell types of marine sponge Hymeniacidon perleve under light and transmission electron microscope. A protocol was produced for identifying, counting archaeocytes under light microscope. Furthermore, we used BrdU incorporation method and PCNA antibody to prove that, as the toti/multipotent cells of sponge, archaeocytes havegreat potential to proliferate.Secondly, this study developed a novel four-step protocol for the purification of archaeocytes from a marine sponge Hymeniacidon perleve: (1) differential centrifugation; (2) selective agglomeration; (3) differential adherence; (4) Ficoll-Vrografin density gradient centrifugation. The final purity of archaeocytes is above 80%. The proliferation potential of the archaeocytes was demonstrated by high levels of BrdU incorporation, PCNA expression and telomerase activity.In a 4-day primary culture, the purified archaeocytes showed a 2.5-fold increase in total cell number. But the proliferation potential of the archaeocytes during sub-culture decreased, probably because of archaeocytes differentiation.As sponge cell populations with different archaeocyte percentage showed different growth protential. Pinacocytes and collencytes are basically two main functional cells with terminal differentiation, so, in this study, the function of the pinacocytes and collencytes in regulating archaeocytes differentiation was investigated. The results showed that when the ration of pinacocytes, collenctyes to archaeocyte was 2:1, the aggregation could differentiate into small functional sponges. Although there was some differentiation in the aggregates of purified archaeocytes, it was hard for them to form functional sponges.Lastly, antibody to the specific protein of sclerocyte, silicatein, was produced. And it could be used by immunocytochemally to mark the sclerocytes before they produce recognizable spicules. Thus a specific method to identifying sclerocytes was established to follow up the differentiation process of archaeocytes to sclerocytes.