The impact of antibiotics on in vitro cell proliferation and primmorph formation in the marine sponge Hymeniacidon perlevis

Details

Supervisors

Rees, Jean-François

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en sciences agronomiques, à finalité spécialisée

Abstract

Sponges are aquatic animals belonging to the basal metazoan phylum known as Porifera, one of the oldest forms of life on Earth, and are essential components of coral reef ecosystems. Recent studies have shown that these primitive filter feeders produce bioactive metabolites derived from their cells or from symbiosis with microorganisms, playing a specific role in their defense against environmental threats. In addition, it has been revealed that these bioactive compounds possess pharmaceutical properties against various diseases. This master's thesis is part of Professor Jean-François Rees's project to develop a methodology for producing living sponges by 3D bio-printing of isolated sponge cells, offering considerable potential for both ecological research and medical applications. This project combines two advances: the optimization of in vitro sponge cell culture and 3D bio-printing. Three-dimensional bio-printing is a promising technique that is revolutionizing advances in medicine and biotechnology. The study focused on optimizing in vitro sponge cell culture using Demospongiae Hymeniacidon perlevis. Optimization was explored through an alternative to the direct use of antibiotics in cell culture, which seem indispensable due to their symbiotic relationship with microorganisms, but which negatively affect the physiology of sponge cell. A pretreatment with antibiotics, combining amphotericin B and rifampicin, reduced culture contamination while promoting increased cell proliferation, thus demonstrating its effectiveness. The effect of cell proliferation was compared between artificial seawater (ASW) and a nutrient-rich complete medium (M199), showing significant proliferation in a nutrient-rich medium compared to ASW, as measured by automated cell counter. However, no significant difference was observed between the same media with or without antibiotics, since the automated cell counter does not take cell viability into account. Using an alternative method combining viability and cell proliferation counts, the MTT assay identified that a 20% dilution of M199 medium was the most conducive to cell proliferation compared with the other dilutions (2% and 100%). The addition of antibiotics to cell culture medium significantly suppressed cell proliferation. Furthermore, diluted media, such as those mentioned above, were used to promote mass growth of non-dissociated sponge explants. However, the five-day culture period applied was not sufficient to observe significant changes, and their regeneration would be enhanced by algae supplementation. This master thesis is a small part of a vast project that points to a definite future in optimizing in vitro cell proliferation in marine sponges, thanks to the conclusive discovery of pretreatment with antibiotics and dilution of a nutrient-rich medium, aided by the effectiveness of the MTT assay on sponge cells. Further research is needed to understand symbiotic microorganisms and optimize growth conditions of sponge explants.