Robust strategies for generating perfusable microvasculature-on-a-chip models

Introduction:: Self-organized microvascular networks (MVNs) have become critical to developing microphysiological models that can mimic the structure and function of complex tissues and organs. These models have been used to study a wide range of biological processes and diseases, such as cancer, inflammation, and drug toxicity. However, the self-organizing nature of this process combined with variations between types or batches of endothelial cells (ECs) often leads to inconsistency or failure to form functional MVNs with physiological morphology and dimension.

Materials and Methods:: To address these issues, we developed several robust strategies to generate perfusable microvasculature-on-a-chip models. Firstly, we utilized immortalized vascular cells and endothelial cells (ECs) differentiated from induced pluripotent stem cells (iPSCs) as stable cell sources. The immortalized vascular cells were generated by exogenously expressing human telomerase reverse transcriptase (hTERT). iPSC-derived ECs were differentiated using a doxycycline-inducible ETS Variant Transcription Factor 2 (ETV2) expression protocol. Secondly, we developed a novel two-step seeding method to generate perfusable capillary-like vessels in vitro. Finally, we applied interstitial flow (IF) to improve vasculogenesis in general. We evaluated microvascular network morphology, perfusability, and permeability using confocal microscopy. We explored EC differentiation, stromal cell markers, and mechanisms of vasculogenesis using immunostaining, flow cytometry, RT-PCR, inhibitor and antibody blocking, and CRISPR-knockout assays.

Results, Conclusions, and Discussions:: The immortalized vascular cells formed functional MVNs even after 20 passages. We identified Thy1-positive lung fibroblasts that express various lumen formation factors and support MVN formation. ECs differentiated from ETV2-iPSCs had a higher yield compared to growth factor-based differentiation protocols. These ETV2-iPSC-ECs were able to form MVNs for personalized tumor-on-chip models to evaluate chimeric antigen receptor (CAR)-T efficiency and for liver organoid vascularization.

Our two-step seeding strategy was designed to form large open lumens at the outer layer region, connecting a narrow diameter MVN in the central region of a microfluidic device, resulting in a fully perfusable, capillary-like MVN. This strategy could be applied to generate skin vasculature models, blood-brain-barrier models, and iPSC-derived blood vessels.

Finally, applying interstitial flow (IF) upregulated matrix metalloproteinase-2 (MMP-2) expression in ECs, leading to improved MVN formation with higher vessel density and greater network perfusability. Using IF and MMP-2 inhibitor, we regulated the morphological parameters of the MVNs while maintaining their vascular permeability and perfusability.

Overall, these strategies have the potential to be widely utilized to boost the development of various organotypic MVNs in vitro and can be incorporated into related bioengineering applications where perfusable vasculature is desired.


Acknowledgements (Optional): :

References (Optional): : Shun Zhang^#, Zhengpeng Wan^#, Georgios Pavlou, Amy X Zhong, Liling Xu, Roger D Kamm*. “Interstitial Flow Promotes the Formation of Functional Microvascular Networks In Vitro through Upregulation of Matrix Metalloproteinase-2.” Advanced Functional Materials, 2022, 32, 2206767.

Zhengpeng Wan, Amy X Zhong, Shun Zhang, Georgios Pavlou, Mark F Coughlin, Sarah E Shelton, Huu Tuan Nguyen, Jochen H Lorch, David A Barbie, Roger D Kamm*. “A Robust Method for Perfusable Microvascular Network Formation In Vitro.” Small Methods, 2022, smtd.202200143.

Zhengpeng Wan^#, Shun Zhang^#, Amy X Zhong, Sarah E Shelton, Marco Campisi, Shriram K Sundararaman, Giovanni S Offeddu, Eunkyung Ko, Lina Ibrahim, Mark F Coughlin, Tiankun Liu, Jing Bai, David A Barbie*, Roger D Kamm*. “A robust vasculogenic microfluidic model using human immortalized endothelial cells and Thy1 positive fibroblasts.” Biomaterials, 2021, 276, 121032.