Timing Matters: Impact of Flow and Hypoxia on Capillary Tube Formation in Bioartificial Fibrin-Based Matrices

Vascularization and perfusion are essential for tissue engineering of large-scale bioartificial tissues and organs. Both fluid flow and hypoxia have been shown to influence capillary tube formation in bioartificial matrices. However, the ideal timing and combination of these two factors for optimizing capillary tube formation in tissue engineering approaches still remains unclear to date. Capillary tube formation was evaluated in 3D flow chamber wells, in which human microvascular endothelial cells (HMVEC) and adipogenous stem cells (ASCs) suspended in fibrin-based tissue matrices were exposed to fluid flow and hypoxia under defined conditions. Here, both factors were either applied alone or in combination, either from the beginning of the incubation period or after initial capillary tube formation on day 3. Capillary tube formation was analyzed by fluorescence microscopy and quantified two times on days 3 and 6. Furthermore, large-scale fibrin matrices surrounded a perfused microchannel under selected conditions. Exposure of HMVEC in fibrin-based matrices to flow and hypoxia alone or in combination immediately from the beginning of the in vitro culture resulted in impaired capillary tube formation compared with static or normoxic culture conditions in all settings. When initiated after initial tube formation under static conditions for 3 days, exposure to physiological fluid shear stress with or without parallel exposure to hypoxia was associated with significantly increased network complexity. The inhibition of capillary tube formation by continued fluid flow could be compensated for by adding exposure to hypoxia after 3 days of initial tube formation. The impact of both fluid flow and hypoxia was highly dependent on the timing of onset as well as their application alone or combined. Both fluid flow and hypoxia were identified as potent tools for regulating capillarization in vascular tissue engineering, but timing and combination are crucial for optimized in vitro tube formation.Impact StatementThe integration of a vascular network providing oxygen and nutrient supply throughout large-scale bioartificial tissues remains a major challenge in tissue and organ engineering. We here provide a timing-specific analysis of the impact of fluid flow and hypoxia on in vitro capillary tube formation with or without static and normoxic preincubation. With this, the ideal timing and combination of flow and hypoxia for capillary tube formation were identified. These insights can be used to refine the perfusion and culture strategies for tissue engineering of vascularized constructs and thus, optimize in vitro prevascularization of large-scale bioartificial tissues and organs.