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99-Metastabil Technetium (^99mTc) is a radiopharmaceutical widely used in skeletal scintigraphy. Recent publications show it can also be used to determine the osteogenic potential of human mesenchymal stem cells (hMSCs) by binding to hydroxyapatite formed during bone tissue engineering. This field lacks non-destructive methods to track live osteogenic differentiation of hMSCs. However, no data about the uptake kinetics of ^99mTc and its effect on osteogenesis of hMSCs have been published yet. We therefore evaluated the saturation time of ^99mTc by incubating hMSC cultures for different periods, and the saturation concentration by using different amounts of ^99mTc activity for incubation. The influence of ^99mTc on osteogenic potential of hMSCs was then evaluated by labeling a continuous hMSC culture three times over the course of 3 weeks, and comparing the findings to cultures labeled once. Our findings show that ^99mTc saturation time is less than 0.25 h, and saturation concentration is between 750 and 1000 MBq. Repeated exposure to <i>γ</i>-radiation emitted by ^99mTc had no negative effects on hMSC cultures. These new insights can be used to make this highly promising method broadly available to support researchers in the field of bone tissue engineering using this method to track and evaluate, in real-time, the osteogenic differentiation of hMSC, without any negative influence on the cell viability, or their osteogenic differentiation potential.