Establishment And Optimization Of In Vitro Cultivation Of Sponge Primmorph: Aggregation, Growth And Metabolism

Marine sponges (Porifera) represent the evolutionary oldest multicellular animals and are rich sources of potent bioactive compounds. They existed prior to the 'Cambrian Explosion' about 580 Million-years ago. There is increasing interest in biotechnological production of marine sponge biomass owing to the discovery of many commercially important bioactive metabolites in this group of animals. One major obstacle for commercialization is the limited availability of sufficient quantities of defined sponge starting material. In vitro sponge cell cultures appear to be very attractive because they are completely controlled and can easily be manipulated for optimal production of the target metabolites. However, this technique is still in its infancy: a continuous cell line has yet to be established. The recent establishment of sponge primmorphs, multi-cellular aggregates from dissociated mixed cell population (MCP), has been widely acknowledged to hold great promise for cultivation in vitro. The aims of this work are to study the mechanisms for cell aggregation,division/propagation, and metabolism in in vitro sponge primmorph culture system; and to further optimize the primmorph culture toward the establishment of a continuoussponge cell line.Primmorph cultures from MCP were established using several marine sponges species, collected from China Oceans and Mediterranean Sea. Methods for qualitative and quantitative characterization of primmorph culture were developed, which included a modified MTT assay for measurement of cell viability and cell growth, a 5-BrdU-incorporation assay for detection of cell proliferation, propagation and metabolism of in vitro primmorph.To identify the key factors affecting primmorph formation and to optimise the technique for developing an in vitro primmorph culture system, a Chinese sponge, Stylotella agminata (Ridley), collected from the South China Sea, were investigated. The size of dissociated cells from S. agminata is relatively small, in the range between 5 and 10 mm. Round shaped primmorphs of less than 100 mm were formed 3 days after transferring the dissociated cells into seawater containing Ca2+ and Mg2+. The microscope studies on the dynamics and kinetics of primmorphformation suggest a tentative mechanism that primmorphs are formed through four stages: amorphous large cell floe, small irregular cell aggregations, large primary cell aggregations and round-shaped primmorphs. Large primary cell aggregations grow via agglomeration/aggregation of both small aggregations and single cells, and finally contacted into round-shaped, regular primmorphs.The effect of various cell dissociation conditions, inoculum cell density, concentration of antibiotics, pH, and temperature was further investigated upon the formation of primmorphs. The time required for primmorph formation, primmorph size distribution, and the proliferating capability were microscopically documented. For a successful formation of primmorphs, it is mandatory to use healthy sponge tissues and a minimum inoculate cell density is required. Culture temperature will need to be adapted to the environmental temperature where the sponges are collected. Further more, the size of primmorphs (average 90-100 m) from S. agminata is much smaller than that of primmorphs from S. dommucula and D. avara(l-3mm), which may be sponge species-dependent. Preliminary data shows sponge cells undergo an initial growth for 3-5days during primmorph formation.The development of a growth medium is one of the essential prerequisites for sponge cells grown in vitro. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was adapted to screen potential nutritional factors in formulating a growth medium for a primary cell culture of Suberites domuncula. In 96-well plates, effects of nutritional factors including glutamine, pyruvic acid, iron citrate, silicon, RPMI 1640 and Marine Broth 2216 were examined on the viable cell density in primary cell culture of S. domuncula after 36 hours of inoculation. Ferric (Fe3+)...