Human bone marrow multipotent mesenchymal stromal cells (hBM-MSC) represent an appealing source of adult stem cells for cell therapy and tissue engineering. Since hBM-MSC are present at low frequency in the bone marrow, in vitro expansion is necessary prior to performing clinical studies. Standard incubator gas tensions corresponding to 20% O2 and 5% CO2 are routinely used for in vitro expansion of hBM-MSC, and it is being overlooked that such conditions do not correspond to the physiological gas tensions found in the bone marrow microenvironment. To explore the effects of different gas tensions, polyclonal hBM-MSC from three donors were expanded in 6 different combinations of O2 and CO2 tensions. Phase contrast microscopy was used to determine cell morphological features, growth curves were made to assess differences in proliferative capability, real-time RT PCR was used to measure gene expression of markers associated with undifferentiated embryonic stem cells, and microarray analysis was performed to assess the global gene expression of the cells. No change in morphology could be observed between hBM-MSC cultured in the different gas tensions. The results from the growth curves indicated that gas tensions did not affect the proliferative capability of the cells. Real-time RT PCR data showed no consistent difference in gene expression between the culture conditions. According to the microarray analysis, there was no significant difference in global gene expression between hBM-MSC cultured in the different gas tensions.
Most current protocols for in vitro culture of hBM-MSC include fetal bovine serum (FBS) as nutritional supplement. When culturing hBM-MSC in FBS, depletion of CD14+ monocytes seems to be necessary in order to avoid monocyte contamination in the cultures. In this study, FBS was replaced by autologous serum (AS). As a part of this thesis, the necessity of depleting CD14+ cells when expanding hBM-MSC in AS was investigated. Only minor contamination of monocytes was observed in the non-depleted cultures, indicating that there is no need to deplete CD14+ cells when expanding hBM-MSC in AS.