Low Oxygen Treatment for the Optimization of 3D Endothelial Cell Tissue Cultures.
Mijailovic, Aleksandar S.
- Currently, the most salient challenge in tissue engineering is the inability to sufficiently vascularize engineered tissues. Without adequate vasculature, most tissues cannot survive due to lack of nutrient and oxygen diffusion. Thus, the therapeutic capability of tissue engineering remains limited. A number of studies have shown that mimicking in vivo oxygen tensions (4%-6%O2) promotes proliferation ... read moreand improves cellular organization of endothelial cells in 2D in vitro cultures (Zhao et al. 2008, Decaris et al. 2009, Zhou et al. 2000). Moreover, one recent study has shown that 5% oxygen pre-treatment increases endothelial cell retention on PET scaffolds under dynamic culture, illustrating the potential of low oxygen culture for engineering vascular tissue grafts (Zhao et al. 2008). In this study, we attempted to engineer a pre-vascularized tissue culture by optimizing oxygen concentration. Through the use of low oxygen (5% O2) environments we aimed to (1) stimulate proliferation and cellular organization of endothelial cell tissue cultures on a 3D porous silk scaffold, and (2) improve cellular retention on 3D silk scaffolds in dynamic cultures. We assessed the potential of low oxygen pre-culture through a series of methods: DNA quantification assays, which were used to monitor cell proliferation; histology and confocal microscopy, which were used to assess cell organization; and real time RT-PCR, which assessed the cells' synthesis of extra cellular matrix (ECM) adhesion proteins, such as CD31, in response to low oxygen. While preliminary DNA quantification results suggested that a 5% oxygen environment improved endothelial cell proliferation compared to 21% oxygen controls, subsequent studies yielded conflicting results. Studies also showed that low oxygen did not significantly affect cell retention in dynamic tissue cultures, and it did not affect CD31 transcript levels, as compared to 21% oxygen controls. Finally, analysis by histology and confocal microscopy did not show improved endothelial organization between oxygen groups.read less