Optimization of cell culture conditions and growth evaluation methods for the demosponge Hymeniacidon perlevis

Details

Supervisors

Rees, Jean-François

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

Sponges are benthic animals living near the sea floor and are fascinating for many reasons. A myriad of molecules derived from sponges and sponge symbionts are used in human medicine as antibacterial, anticancer or antibiotic agents. The need for large amount of sponges for preclinical studies cannot be satisfied by harvesting from nature as this could cause severe damage to sponge population. As a solution, sponge cell lines could produce enough material for fundamental studies. Moreover, the incredible body and cellular plasticity of sponges are due to remarkable totipotent cells capable of transdifferentiation into almost every other cell type: the archaeocytes. A promising alternative to increase the proliferating capacities of the starting cell suspension could be to enrich it with totipotent stem cells. For this to happen, proper optimized cell culture conditions and method evaluating cell growth must be developed. This master thesis is part of a project led by Professor Jean-François Rees and aims to develop a methodology to produce living sponges through the bioprinting of isolated sponge cells. The globally distributed marine sponge Hymeniacidon perlevis has been chosen as the first candidate for the development of a 3D culture model. Experiments started on cryopreserved Hymeniacidon perlevis sponge cells which conserved their aggregation properties without the addition of antibiotics and antimycotics in a medium deprived of nutrients, artificial seawater (ASW). Cryopreserved sponge cells were cultured in OpM1 and M1, two media developed to establish the first continuous cell line for Geodia barretti. Moreover, cells were also cultured in M199, a commercially available culture medium and a control consisting of ASW. Prestoblue assay was used to test the resulting cell metabolic activities every 24-hours of culture. Results seemed to show that sponge cells were not sufficiently active to provide enough signal to be different from background signal after the first 24-hours of culture. Moreover, results tended to show that cryopreserved cells cultured during 96-hours in OpM1 exhibited higher metabolic activity compared to M1, M199 and control medium ASW. In an attempt to go a step further, a second set of experiments was carried out on freshly dissociated archaecoyte dominant cell population (ADCP) and mixed cell population (MCP) to compare their proliferating capabilities through different methods. To do so, an effective method leading to the enrichment of a freshly dissociated cell suspension in archaeocyte using Percoll gradient density was successfully developed allowing to obtain ADCP. Flow cytometry analysis with optimized parameters has been performed on ADCP and MCP to attempt evaluating cell density and seemed to show that fresh sponge cells suspension cultured in OpM1 during 96-hours had the highest cell density. However, cell counting through optical microscopy has been proven to be difficult and inadequate to properly evaluate cell density. Another method developed was BCA assay aiming to evaluate cell proliferation through cellular protein content evaluation. Initial experiments revealed the necessity for optimizing the volume of the cell lysis solution and conducting proper sponge protein calibration for Hymeniacion perlevis cells specifically. Numerous questions are yet to be addressed to understand the underlying cell proliferation mechanisms of Hymeniacidon perlevis to effectively develop optimized cell culture conditions and growth evaluation methods.