Sponges (phylum porifera) are among the oldest Metazoa and considered critical to understanding animal evolution and development. They are also the most prolific source of marine-derived chemicals with pharmaceutical relevance. Cell lines are important tools for research in many disciplines, and have been established for many organisms, including freshwater and terrestrial invertebrates. Despite many efforts over multiple decades, there are still no cell lines for marine invertebrates. In this study, we report a breakthrough: we demonstrate that an amino acid-optimized nutrient medium stimulates rapid cell division in 9 sponge species. The fastest dividing cells doubled in less than 1 hour. Cultures of 3 species were subcultured from 3 to 5 times, with an average of 5.99 population doublings after subculturing, and a lifespan from 21 to 35 days. Our results form the basis for developing marine invertebrate cell models to better understand early animal evolution, determine the role of secondary metabolites, and predict the impact of climate change to coral reef community ecology. Furthermore, sponge cell lines can be used to scale-up production of sponge-derived chemicals for clinical trials and develop new drugs to combat cancer and other diseases. Sponges (Phylum Porifera) are key components of many benthic marine ecosystems. There are more than 9,000 described species that occur worldwide, from the intertidal to the deep sea 1. Among the oldest metazoans, sponges have evolved a variety of strategies to adapt to different environments. Because they are sessile as adults, they have evolved sophisticated chemical systems for communication, defense from predators, antifoulants to prevent other organisms from growing over them, and to prevent infection from microbes filtered out of the water 2,3. These chemicals interact with molecules that have been conserved throughout evolutionary history and are involved in human disease processes, for example, cell cycling 4 , immune and inflammatory responses 5 , and calcium and sodium regulation 6,7. Vertebrate, insect, and plant cell lines are important tools for research in many disciplines, including human health, evolutionary and developmental biology, agriculture, and toxicology. Although cell lines have been established for freshwater and terrestrial invertebrates (e.g., Hydra, Caenorhabditis), and long-term (>1 month) primary cultures have been reported for cells derived from tissues of the cnidarian Anemonia viridis and the shrimp Penaeus 8,9 , attempts to establish cell lines from marine invertebrates have been unsuccessful 9-11. Marine sponges, including some of the species in this study, are the source of thousands of novel chemicals with pharmaceutically relevant properties 12-14. Supply of these chemicals is a bottleneck to development of sponge-derived drug leads: wild harvest is not ecologically sustainable, and chemical synthesis is challenging due to the complexity of many of the bioactive chemical compounds. In vitro production has been proposed as an option...

Production of sponge-derived bioactive compounds in vitro has been proposed as an alternative to wild harvest, aquaculture, and chemical synthesis to meet the demands of clinical drug development and manufacture. Until recently, this was not possible because there were no marine invertebrate cell lines. Recent breakthroughs in the development of sponge cell lines and rapid cell division in improved nutrient media now make this approach a viable option. We hypothesized that three-dimensional (3-D) cell cultures would better represent how sponges function in nature, including the production of bioactive compounds. We successfully cultured sponge cells in 3-D matrices using FibraCel® disks, thin hydrogel layers, and gel microdroplets (GMDs). For in vitro production of bioactive compounds, the use of GMDs is recommended. Nutrients and sponge products rapidly diffuse into and out of the 3-D matrix, the GMDs may be scaled up in spinner flasks, and cells and/or secreted products can be easily recovered. Research on scale-up and production is in progress in our laboratory.