Results 24 Fig. 8: Lipid content of adipogenic differentiated hMSCs was measured 21 days after adipogenic induction. Results are shown in relative fluorescence units (RFU). Hypoxic cultured cells showed a significant, two-times higher lipid content compared to normoxic cultured cells (p=0.0104). Undifferentiated cells showed significantly less RFU compared to induced groups (Normoxia: p=0.0046, Hypoxia: P=0.001). n=2, r=3, Mean & SD. 3.1.3. Surface receptor expression A flowcytometry analysis was performed to identify the stem cell characteristics in accordance to the statement of the International Society for Cellular Therapy. The positive marker CD73, CD90 and CD105, which should be higher expressed than 95%, were expressed to 99.9%, 100.0% and 100.0% for normoxic cells and 100.0%, 99.9% and 99.9% for hypoxic cells respectively. Negative markers CD14, CD19, CD45 and HLA-DR, which showed to be lower expressed than 2%, were consistently low expressed to 0.67%, 0.43% 0.77% and 0.62% for normoxic cells and 0.50%, 1.27%, 1.42% and 0.54% for hypoxic cells. Figure 9 shows the fluorescence intensity histograms and the corresponding isotype controls for each CD marker and culture condition.
Results 25 Fig. 9: Exemplary cluster of differentiation (CD) expression acquired by flowcytometry analysis for normoxic and hypoxic cultured hMSCs. Isotype controls are shown in red and CD markers are blue. There was no difference between each group. A: Cells in both culture conditions were positive for CD73, CD90 and CD105, B: Cells in both culture conditions were negative for CD14, CD19, CD45 and HLA-DR (A & B, normoxia: upper row, hypoxia: lower row). 3.2. Biological cell characteristics To better understand the influence of different oxygen levels in cell culture conditions, data of biological characteristics of hMSCs were raised. Therefore, cumulative population doublings, population doubling time, metabolic activity, shape description parameters and cell volume were measured. 3.2.1. Cumulative population doubling Measurement of the cumulative population doublings (cum PD) allows the direct comparison of the total doublings of the entire cell population. The cum PD was