Results 28 Fig. 12: WST-Assay to assign the metabolic activity of normoxic and hypoxic cultured hMSCs. Normoxic stem cells were set to 100%. Compared to the control group (red, 100.0 ± 3.2%), hypoxic cultured cells showed a significant higher metabolic activity (blue, 136.9 ± 37.05%, p=0.0012). Experiment was repeated three times in quintets. Mean & SD. 3.2.3. Cell morphology After the typical initial colony forming of primary cell extraction (data not shown), cells in both culture conditions were imaged by phase contrast microscopy over the entire culture period. Figure 13 shows a qualitative overview of all four cell culture quarters. The quarters were set for each culture condition individually, since there was a time slice difference of 12 days until cells went into senescence. Normoxic cultured cells went into senescence after 52 days. Consequently, quarters for evaluation of morphological changes in Normoxia were set to 4, 16, 34 and 52 days respectively. The time until cells within Hypoxia went into senescence was 64 days. Thus, quarters were set for 4, 24, 44 and 64 days. According to the criteria for subpopulations of hMSCs as described in 2.2.3., cells were grouped into rapidly self-renewing (RS), spindle-shaped (SS), flattened (FC) and flattened spindle-shaped (FSS) cells. In the first quarter (4 days after start of cum PD) cells in both culture conditions displayed a homogenous morphology with a large portion of RS and SS cells. During the second (16 days) and third (34 days) quarter in regard to the normoxic cultured cells significantly more FC cells were detectable, while RS and SS cells continuously decreased. However, hypoxic cultured cells maintained their rapidly self-renewing morphology over the second (24 days) and third (44 days) quarter with a majority of RS and SS like cells. During the last quarter, normoxic (52 days) and hypoxic (64 days) cells changed their morphology towards a nearly homogeneous FC population and went into senescence.
Results 29 Fig. 13: Exemplary phase contrast images of normoxic and hypoxic cultured hMSCs. In the first quarter (4 days in culture) cells had a comparable distribution of the different subpopulations for both settings. The fraction of FC (*) cells continuously increased in normoxic conditions over the next quarters (2.Q: 16 days, 3.Q: 34 days in culture) until cell population went into senescence after 52 days (4.Q). Hypoxic cells instead showed a homogenous morphology with an extent of RS (§) and SS (#) cells over the second (24 days) and third (44 days) quarter. In the last quarter, directly before senescence, hypoxic cultured cells also displayed an increased fraction of FC cells. Bar: 200 µm.