Sponge cell culture

Marine sponges are a rich source of bioactive compounds with pharmaceutical potential and are the most prolific source of newly discovered bioactive compounds with more than 7,000 novel molecules discovered in 40 years. Despite its enormous potential, only a few sponge-derived bioactive compounds have been successfully developed into products. A major obstacle is the lack of sufficient supply of biological material for preclinical and clinical development. An option to supply materials for early drug development is production by the sponge, sponge tissue, sponge cells and/or the sponge symbionts. The main challenge with this is to establish generic techniques for small-scale production of sponge biomass. This thesis focuses on gaining insight in cell proliferation and cell death of sponge cell cultures and making the first step towards a continuous sponge cell culture for biotechnological purposes, such as the production of bioactive compounds. In <strong>chapter 2</strong> &ldquo;Cultivation of sponges, sponge cells and symbionts: achievements and future prospects&rdquo;, we analyzed the state of the art for cultivation of whole sponges, sponge cells and sponge symbionts for biotechnological purposes and we elaborate on approaches to overcome bottlenecks, including transformation of sponge cells and using media based on yolk. In <strong>chapter 3</strong> &ldquo;Cell cycle analysis of primary sponge cell cultures&rdquo;, we discussed the development of a flow cytometric cell cycle analysis method to measure the proliferative state of sponge cells, in combination with a caspase assay to detect apoptosis. These methods allowed for a quick determination of the proliferative status of a sponge cell population. We analyzed the cell cycle distribution of five different species (Haliclona oculata, Haliclona xena, Dysidea avara, Axinella polypoides andXestospongia muta) from different locations to compare the proliferative status at the start of the culture. In addition, we measured the cell cycle distribution and caspase activities of cells from H. oculata during cultivation to study the change in distribution of cells over time. Subsequently in <strong>chapter 4</strong> &ldquo;Toward development of a sponge cell line: comparison of cell proliferation in sponge cell and tissue cultures&rdquo;, we used the developed methods from chapter 2 to compare culture conditions of two Mediterranean sponges (D. avara and Crambe crambe) and one Dutch sponge (H. oculata). We hypothesized that in vitro tissue culture will be a more favorable culture condition than cell culture, because the dissociation process is avoided, meaning the cells are not exposed to shear stress and the cell-cell contacts are retained. As a control we also included tissue culture in the sea (in situ) to test, in case of lack of growth, whether this is due to the cutting of the sponge into tissue pieces or to environmental conditions that are different in the sea compared to the in vitro culture. In <strong>chapter 5</strong> &ldquo;Methods for insertion and expression of heterologous genes in sponge cells&rdquo;, we made the first step towards immortalization of sponge cells by comparing different methods for the insertion and expression of heterologous genes in sponge. We tested three gene delivery systems, lipofection, particle bombardment and viral transduction, in juveniles of the freshwater sponge Ephydatia fluviatilis, and lipofection was tested in primary cell cultures of the marine sponge H. oculata. Juveniles of E. fluviatilis were chosen as a model sponge for this research, because they are able to proliferate in vitro. To visualize transfection efficacy, we used the green fluorescent protein (GFP) reporter gene. To test promoter recognition by sponge RNA polymerase, we tested the cytomegalovirus (CMV) and OpIE2 promoter, which are generally used for transfection of mammalian cells and insect cells, respectively. Finally in <strong>chapter 6</strong> &ldquo;Genomic based approach for sponge cell culture&rdquo;, we provided an outlook on how recent developments in the field of genomics and transcriptomics can help us to gain more insight in sponge growth and death and consequently obtain a continuous sponge cell line