In vitro, pre-vlinical & commercial evaluation of a novel xeno- & serum-free culture system for production of human bone marrow derived mesenchymal stem cells for bone regeneration
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The therapeutic potential of mesenchymal stem cells (MSCs) is recognised in treating a wide range of debilitating diseases. For clinical-scale manufacturing, serum-free and xenogenic-free media formulations have been proposed as alternatives to the use of foetal bovine serum (FBS), an undefined product with significant safety concerns. Previously, the orthobiologics laboratory had developed a novel xeno- and serum-free media formulation for the isolation of bone marrow-derived MSCs. The overall goal of this project was to identify the optimal cell culture conditions for both the isolation and expansion of bone marrow derived mesenchymal stem cells for orthopaedic use. This included assessing the effect of varying the oxygen tension during culture and the use of the novel xeno- and serum-free (SF) medium. Specifically, this was determined by assessing the efficacy of SF medium-isolated and cultured bone marrow-derived MSCs for their ability to contribute to bone repair in pre-clinical orthotopic models of bone regeneration namely a mouse ectopic model of bone formation and a rat femoral critical size bone defect model. These cells were compared to conventional FBS-isolated cells to determine if the novel SF medium was a suitable alternative for the manufacture of MSCs. Subsequently, evaluation of the SF medium in comparison to commercially available serum-free media was performed and the medium’s functionality in a 3D macrocarrier bioreactor system for the manufacture of MSCs was also performed. The specific aims of this PhD were as follows; In vitro characterisation of xeno and serum-free MSCs (MSC-SF) and serum-cultured MSCs (MSC-SC) in hypoxia (2% O2) and normoxia (21% O2) were assessed in chapter 3. SF MSCs isolated and cultured in hypoxia underwent increased proliferation in comparison to their SC counterparts whiles maintaining their tri-lineage differentiation potential. In addition, hypoxia-cultured SF MSCs demonstrated an increased chondrogenic potential in comparison to normoxia-cultured SF MSCs which had an increased osteogenic phenotype. These data indicated differences in the phenotype of the cells that may alter the efficacy of these cells to repair bone. In chapter 4, SF and SC MSCs cultured in either hypoxia or normoxia were implanted subcutaneously on an osteoconductive biomaterial in CD1-nude mice for 8 weeks and bone formation was assessed. No difference was observed between the various treatment groups. In addition, no difference in level of bone tissue was observed between treatment groups and vehicle control which received a cell-free material. This data indicated that bone formed was due to recruitment of endogenous cells to the implants. To assess the ability of SF MSCs to repair bone in a clinically relevant model of bone repair, MSCs were implanted into a rat femoral critical size bone defect model for 8 weeks and their bone repair ability was assessed in vivo and ex vivo (Chapter 5). Micro-computed tomography (µCT) analysis of bone regeneration was assessed at 4 and 8 weeks. Subsequently, histological analysis of bone repair was also assessed. These data indicated superior bone repair in groups which received SC MSCs cultured in normoxia with minimal repair in groups which received SF MSCs. Consequently to this, in vitro comparison of the SF media with commercially available media was performed (Chapter 6). Here, equivalent growth kinetics were observed in all media formulations. Increased differentiation potential was observed in commercial media groups, specifically Xuri produced by GE Healthcare and Mesencult produced by Stemcell Therapeutics. Superior production of pro-angiogenic factors were observed in SF medium groups. SF and Xuri media met the international society for cell therapy (ISCT) guidelines for surface marker profile of MSCs while Mesencult-MSCs maintained expression of HLA-DR. The ultimate success of the SF media will depend on its ability to function in scalable processes for large scale production of cell therapies. In chapter 7, the function of the SF medium in a 3D spinner-flask bioreactor system was assessed and demonstrated that SF MSCs maintained their growth kinetics in addition to maintaining their tri-lineage differentiation potential when cultured in the bioreactor system. Together, these data suggest that the SF medium is a suitable alternative to the large scale production of bone marrow-derived MSCs for cell based therapies.
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