Développement d'un milieu sans sérum et d'un procédé à grande échelle pour la prolifération de cellules humaines précurseurs du muscle
|Advisor:||Garnier, Alain; Tremblay, Jacques-P.|
|Abstract:||Duchenne muscular dystrophy (DMD) is a genetic disease affecting one boy out of 3500. It leads to progressive and irreversible muscle degeneration. Currently, there is no cure for this disease and no therapy allows an efficient and safe treatment. Cell therapy consists in injecting healthy human muscle cells, myoblasts, into the muscles of DMD patients. Promising results have been obtained in clinical trials using this approach. However, some problems need to be overcome. Particularly, the original culture medium used to expand myoblasts contains Foetal Bovine Serum (FBS). FBS is an undefined product derived from animal, which can be contaminated with bacteria, viruses or prions. The possibility of harmful consequences for the patients hampers the acceptability of the therapy, so FBS must be replaced by a mixture of defined factors such as specific recombinant cytokines. Moreover, the production processes are currently inappropriate to meet the demand for muscle cells. Therefore, this project aims to develop a serum-free medium for the in vitro proliferation of muscle cells and a large-scale process for the production of those cells. A multi-step method was used to develop the serum-free medium. It is divided into three main steps: a) to build a panel of potential factors from a screen of the literature followed by the detection of more than 100 receptors and autocrine factors using RT-PCR, b) to test those factors in culture using statistical design of experiment (DOE) allowing to verify individual and synergistic effects and c) to add the factors that generate beneficial response to the culture medium. Those steps are followed until a cellular response comparable to the culture in standard medium is obtained. To achieve the scale-up objective, the project was limited to the selection of microcarriers that allowed the proper adhesion of myoblasts. The potential factors identified in the first stage, together with additional ones taken from the literature, formed a panel of 9 basal culture media and 72 additives that have been tested by means of DOE. At the end of this process, a serum-free medium, LOBSFM, was developed. To our best knowledge, it is the only existing efficient serum-free medium for myoblast proliferation and a patent has been obtained to protect its use. It allows a specific expansion of myoblasts (~80% myoblasts, verified by immunostaining) comparable to a standard medium containing 15% FBS over a 60-day culture period. Moreover, myoblasts kept their ability to form muscle fibers. Porous microcarriers Cytoline2 allowed a final cell concentration of 1.5E6 cells/mL, comparable to cell expansion in static culture plate. The pores protected the cells against mechanical stresses while the cell recovery remained easy.|
|Document Type:||Thèse de doctorat|
|Open Access Date:||24 April 2018|
|Collection:||Thèses et mémoires|
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