Within the framework of the BLUES project, Cellex has focused on adapting and optimizing the BioAxFlow platform for the culture of marine invertebrate cells, particularly those derived from sponges.

These organisms are of growing interest due to their capacity to produce bioactive molecules, although harvesting those from their natural sources poses a risk for oceans-ecosystem maintenance, hence developing a sustainable in vitro cultivation procedure would be desirable.

To address this, Cellex developed a dedicated scaffold holder to be placed inside the BioAxFlow chamber. This holder was engineered specifically to accommodate scaffolds composed of shark collagen, a substrate upon which these sponges normally grow in nature, ensuring mechanical stability and proper fluid dynamics during perfusion-based culture. The holder was tailored to fit the unique configuration of the modular bioreactor while maintaining compatibility with its perfusion systems.

The key role of marine sponges in scientific research

Sponges are among the oldest metazoans and are crucial to research, as they are the most prolific source of pharmaceutically relevant marine-derived chemicals.

Indeed, sponges contain structural components with potential applications for human health, from tissue engineering (where they can be used as a scaffold to grow bone implants) to the development of new drugs against cancer and viral, bacterial and fungal infections.

However, a problem arises: how can biomass be obtained from sponges without altering entire ecosystems?

Indeed, the massive isolation of these animals for extracting bioactive compounds would have a disastrous impact on the environment. That is why several alternative proposals have been put forward in recent decades.

The first was the chemical synthesis of sponge metabolites, which unfortunately turned out to be not feasible because of the complex chemical structures of the compounds, which make synthesis difficult or expensive.

The transfection of genes involved in the production of certain bioactive substances into a culturable microorganism has also been explored; however, the large number of genes involved in the metabolism of sponges complicates this process considerably.

Alternatively, various methods for sponge culture have been used, with varying success, including:

• Mariculture: based on techniques learnt from the experience of mariculture with swimming sponges, where different species of sponges were cultivated in their natural habitat;
• Cultivation of sponge explants under controlled or semi-controlled environmental conditions (closed or semi-open systems);
• In vitro cultures: cultures of explants, primordia or dissociated cells under fully controlled conditions.

Although promising, even in sponge cell culture in vitro researchers encountered considerable difficulties, which were attributed to inaccurate systems for measuring cell proliferation, inadequate culture conditions for growth, and

the sponge cells being in a non-proliferative state at the start of the culture already.

For decades, this was a major obstacle that blocked research. Then, in 2023, the first continuous marine sponge cell line originating from Geodia barretti was created.

This was the starting point for the BLUES project.

The first results of the BLUES project

The BLUES project aims at taking advantage of cutting-edge technologies to develop marine invertebrate cell lines, to be used for getting valuable and unique bioactive compounds through industrial bioprocesses.

Among the phyla that are being used to derive individual cell lines, there are sponges (Porifera), corals (Cnidaria), sea cucumbers (Echinodermata), and ascidians (Chordata). Each of these organisms contributes to the rich tapestry of marine biodiversity and holds promise for sustainable biotechnological advancements.

Initial proof-of-concept experiments have been conducted using sponge cells (Geodia barretti). As we said, Geodia barretti’s first continuous cell line has been generated at the University of Wageningen, posing a crucial milestone for what is planned next during the project.

Two CELLEX bioreactors (110 mL working volume each) were used to test two distinct scaffold materials: polylactic acid (PLA) and a composite of shark collagen with hydroxyapatite (S-HA), the latter developed at the “Universidade do Minho”. These scaffolds were inserted into the bioreactors and colonized by Geodia cydonium cells, a marine sponge species known for its high regenerative capacity and unique biomineralization properties, thanks to researchers from Università di Genova and University of Wageningen.

These preliminary trials, still ongoing, aim to evaluate the colonization efficiency and viability of sponge cells on a shark collagen-hydroxyapatite composite (S-HA) scaffold, developed by the University of Minho, selected for its improved biofunctionality.

While the results are still at an early stage, the integration of custom scaffold holders into BioAxFlow has enabled the controlled exposure of marine sponge cells to dynamic culture conditions, a key step toward scalable in vitro systems.

The technical versatility of BioAxFlow, enhanced by Cellex’s scaffold holder innovation, lays the foundation for marine bioprocess development, supporting future efforts in sustainable production of high-value compounds from marine invertebrates.

Sources

Breakthrough in Marine Invertebrate Cell Culture: Sponge Cells Divide Rapidly in Improved Nutrient Medium, Megan Conkling, Kylie Hesp, Stephanie Munroe, Kenneth Sandoval, Dirk E. Martens, Detmer Sipkema, Rene H. Wijffels & Shirley A. Pomponi, 2019

Cell cycle analysis of primary sponge cell cultures, Klaske J. Schippers, Dirk E. Martens, Shirley A. Pomponi, René H. Wijffels, 2011

Cell culture from sponges: pluripotency and immortality, Sònia de Caralt, María J. Uriz, René H. Wijffels, 2007

First continuous marine sponge cell line established, Hesp, K., van der Heijden, J.M.E., Munroe, S. et al., 2023

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