Growth and metabolism of mesenchymal stem cells cultivated on microcarriers

Mesenchymal stem cells, MSCs, are a great potential source for clinical applications in the field of tissue regeneration. Although MSCs can be isolated from several tissues of the human body, e.g. the bone marrow, the tissues does not contain clinically relevant amounts of MSCs for cell therapeutic use. This dissertation describes the expansion and metabolism of MSCs in a microcarrier-based bioreactor system. Growth and metabolic experiments in T-flasks using MSCs from goat, rat and human donors showed that there is a species dependence on growth rate and metabolic utilization of glucose. Moreover, it was shown that MSCs can tolerate relatively high lactate concentrations before growth inhibition occurs, while ammonia inhibits MSC growth at low levels that are faced during culture. We showed the possibility to expand goat and human MSCs on Cytodex 1 microcarriers in a spinner flask culture. By optimizing the feeding regime based on cellular metabolism, cell growth on microbeads was improved and clinically relevant numbers of MSCs per cm2 were reached, while maintaining stem cell characteristics and multipotency. In addition, we showed that when controlling the pH at 7.2-7.3 and maintaining the oxygen tension at hypoxic levels of 3-4% oxygen in a stirred vessel bioreactor, cell expansion and cell viability was improved, and a better maintenance of human stem cell multipotency, with regard to chondrogenesis, was obtained. Moreover, we found that human MSC metabolism shifted to a higher rate of glycolysis to generate energy for growth and cellular maintenance when expanded at low oxygen tensions. A detailed insight in the amino acid metabolism of the human MSCs expanded at a low oxygen tension in a controlled stirred culture is presented. A batch culture for 6 days showed no limitations in amino acid concentrations. In addition to cell culture characteristics, in vivo studies with MSCs expanded in a microcarrier-based bioreactor system were initiated to examine the preservation of stem cell characteristics during expansion in vivo. The Appendix chapter described an efficient method of MSC enrichment and expansion of porcine MSCs mimicking the clinical situation.