Dawn of a new era in sponge biotechnology

Sponges are recognized as a treasure trove of potential new drugs, as they produce many biologically active compounds to interact with their surroundings and protect themselves. Despite all efforts and some promising results, attempts to develop sponge cell lines to produce compounds in a controlled environment were ultimately unsuccessful.

In Chapter 2 we reported a long-awaited breakthrough: cells of 9 sponge species divided rapidly in amino acid-optimized nutrient medium M1, based on mammalian cell culture medium 199 (M199). Members of the genus Geodia, G. neptuni, Geodia sp., and G. barretti, showed the least individual variation. The Geodia spp. were subcultured 3 - 5 times over a period of 21 - 35 days and reached an average total of 6 population doublings. These finite cell lines represented the first real leads to develop continuous marine sponge cell lines.

In Chapter 3 we established that G. barretti cells proliferated even more rapidly and to a higher density when cultured in OpM1 medium, the successor of M1 with added growth factors, lipids, vitamins, and other nutrients. G. barretti cells of 3 individuals could double nearly 100 times in OpM1, compared to 5 doublings cells of the same individuals reached in M1. These results brought us one step closer to sponge cells producing biopharmaceuticals at industrial scale.

In Chapter 4 we compared genes expressed by G. barretti cells in fragments of an intact sponge, after being dissociated and cryopreserved, and when cultured in OpM1 medium. Telomerase reverse transcriptase, the gene that determines the number of times the genome can be replicated, was expressed in all samples, providing further evidence that sponge cells may be naturally immortal. Cultured cells reorganized their cytoskeleton, down-regulated genes needed to adhere to the extracellular matrix, synthesized more proteins and lipids, phagocytosed less and broke down fewer lipids and glycogen reserves for energy, likely favoring soluble energy sources in the medium. These results gave us unprecedented insight into the inner workings of sponge cells.

In Chapter 5 we demonstrated that cultured G. barretti cells can be genetically modified with the CRISPR/Cas12a system. We used CRISPR/Cas12a to introduce a double-stranded break and insert a scrambled DNA sequence in the G. barretti 2',5'-oligoadenylate synthetase gene through homology-directed repair by providing a short single-stranded DNA donor. A longer double-stranded DNA donor was used to insert and express a blue fluorescent marker gene, although efficiencies were low. Introducing longer constructs as single-stranded donors may allow more efficient gene editing. Our results represent an important step towards developing a molecular tool box for sponge cells.

Chapter 6 reflects on what we have learned in the previous chapters, which questions have arisen from this new knowledge, and what next steps should be taken. Now that we can culture sponge cells consistently and possibly continuously, many new lines of research have opened up. Each new line of research will present a new set of challenges, but we now have the required tools and knowledge to overcome these challenges. It is the dawn of a new era in sponge biotechnology.