In Vitro Culture Of Gonad Cells, Hemocytes And Germline Stem-Like Cells From Ascidian (Ciona Savignyi)

Ascidian(Ciona savignyi)is an important model animal for studies of evolution and comparative biology.The success to culture cells from ascidian will greatly to contribute in vitro approach as to study gene expression and regulation.The purpose of this study is to find out and optimize the culture conditions for the growth of ascidian cells and establish immortalized ascidian cell lines in vitro.The tissues used for primary cultures are gonad and hemocytes.The media selected are L-15,M199,DMEM and RPMI1640.The optimal culture conditions for gonad cells in vitro are:M199 medium containing 10%FBS.It also is found that L-15 supplemented with 10%fetal bovine serum best supports the growth of ascidian hemocytes.And the optimal temperature and pH for the growth of ascidian cells is 20℃and 6.8,respectively.The identification and prevention of bacterial contamination in cell culture were investigated.Two bacterial strains that were contaminated in cell culture were isolated from the bacterial contamination samples.The detected bacterium were isolated and identified as the members of by 16S rDNA sequencing.Two bacterial strains were gram-negative bacterium.In order to control the microbial contamination in cell culture,three antibiotics mixture were compared.It is shown that gentanycin was efective to prevent bacterial contamination.However,primary culture of gonad cells and hemocytes are difficult to proliferate and has a short lifespan in vitro.Germline stem(GS)cell is an adult stem cell population with the capability of self-renewal and differentiation throughout life. GS cell culture may provide a new approach for the successful development of cell cultures from marine invertebrates.In the present study,we report that GS-like cells were derived from the adult gonads of Ciona savagnyi and embryonic stem(ES)-like cells would appear in the cultures during the initiation of gonad cells cultures.These ES-like cells were similar to those previously described pluripotent stem cells in morphology and gene expression and had a potential ability to differentiate in vitro, suggesting that ascidian gonads might retain the ability to generate pluripotent cells.A novel invertebrate TNF ligand was identified and characterized in Ciona savignyi.The CsTL cDNA consisted of 995 nucleotides and encoded 251 amino acids. Expression studies demonstrated that the transcription of CsTL mRNA is higher in gonads tissues.The Recombinant CsTL protein expressed using a baculovirus expression system showed potential cytotoxic activity in L929 cells but no effection on gonad cells.