High-resolution, non-destructive imaging with micro-computed tomography (μCT) enables in situ monitoring of tissue engineered bone constructs. However, it remains controversial, if the locally imposed X-ray dose affects bone development and thus could influence the results. Here, we developed a model system for μCT monitoring of tissue engineered bone-like constructs. We examined the in vitro effects of high-resolution μCT imaging on the cellular level by using pre-osteoblastic MC3T3-E1 cells embedded into three-dimensional collagen type I matrices. We found no significantly reduced cell survival 2 h after irradiation with a dose of 1.9 Gy. However, 24 h post-irradiation, cell survival was significantly decreased by 15% compared to non-irradiated samples. The highest dose of 7.6 Gy decreased survival of the pre-osteoblastic MC3T3-E1 cells by around 40% at 2 days post-irradiation. No significant increase of alkaline phosphatase (ALP) activity at 2 days post-irradiation was found with a dose of 1.9 Gy. However, ALP activity was significantly decreased after 7 days. Using our model system, the results indicate that μCT imaging with doses as low as 1.9 Gy, which is required to obtain a reasonable image quality, can induce irreparable damages on the cellular level.