(I-338) Optimizing perfusable and stable 3D microvascular network within human bone marrow-on-a-chip devices by using alternate endothelial cell sources

Three-dimensional microfluidic organ-on-a-chip models of organs such as the human bone marrow (hBM) contain vasculature and interconnected cellular layers which allow in vitro study of microphysiological environments and pathologies1. Fully functional vasculature is needed to transport cells, nutrients, and waste throughout the model and is composed of endothelial cells (ECs) and other stromal cells such as fibroblast and mesenchymal stromal cells (MSCs). Various kinds of ECs have been used for this network throughout the past, however these have been discarded due to limited availability and high variability from week-to-week since the cells were donor and age dependent. This led to a need for alternate sources for generating ECs which would form a stable and perfusable microvascular network within a human bone marrow (hBM)-on-a-chip model and display less weekly variability. Two potential options were considered: blood outgrowth endothelial cells (BOECs) and immortalized human umbilical vein endothelial cells (imHUVECs). BOECs are endothelial progenitor cells that can be isolated from human blood and cultured to induce EC differentiation in vitro2. imHUVECs are HUVECs which have been genetically manipulated to maintain phenotypic characteristics and proliferate indefinitely3.

BOECs were cultured in Endothelial Growth Medium supplemented with fetal bovine serum in collagen-coated flasks. Once proliferated, the cells were stained with antibodies for CD31, CD45, and CD68 for characterization. imHUVECs and blue fluorescent protein-expressing (BFP-imHUVECs) cells were obtained from Dr. Roger Kamm's lab at MIT and cultured in VascuLife Media.The hBM-on-a-chip device was fabricated using a bottomless 96-well plate, one layer of Polydimethylsiloxane (PDMS) and one layer of film. ECs and Normal human lung fibroblasts (NHLFs) or mesenchymal stromal cells (MSCs) as supporting stromal cells were loaded in a fibrin-collagen I hydrogel in the central channel. Additional NHLFs were seeded into the outer channels in order to provide additional support to the vasculature formation. Finally, the media channels were hydrated with medium supplemented with vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANG-1).  Media conditions and the seeding densities of the various primary and supporting cells were varied to assess which conditions led to optimal development of microvascular networks. The BM-on-a-chip devices were imaged daily and were fixed and stained for CD31 at experimental endpoints.

Daily microscopic imaging showed that the BOECs do not proliferate or colonize adequately. Furthermore, staining for CD31, CD45, and CD68 showed that these cells had no clear phenotypic characterization, and thus were unfit for loading and vascular formation. On the other hand, the imHUVECs demonstrated exceptional proliferation and vascular network formation in preliminary results. In fact, by Day 3 of culture, these cells had grown from 2.5e5 cells total to 2e6 cells total. On Day 13, the cells were loaded into hBM-on-a-chip devices with conventional HUVECs as a control. Within four days in the devices the imHUVECs reached 35% coverage, with 50% coverage being the minimum threshold for vasculature coverage in the devices to be deemed stable. To reach and exceed this baseline, the seeding density of the imHUVECs was altered, and it was found that a lower density produced greater coverage, although the perfusability was still not optimal. Therefore, the growth medium was altered: VascuLife, EGM with standard concentrations of VEGF and ANG-1, and EGM with increased VEGF and/or ANG-1. Among these conditions, it was found that EGM with increased VEGF and ANG-1 produced greater vascular branching and vessel diameters closer to that which is required for transport of nutrients, waste, and cellular components through the device. Next, the seeding density and identity of the supporting cells will be changed in an attempt to determine their effects on the development of vasculature. Our results so far have demonstrated that imHUVECs have the potential of replacing HUVECs as the gold standard for the source of ECs in the BM microenvironment. If so, this would provide an accessible method for creating patient-specific treatments and biomimetic models which do not show experimental or donor variability and can be reproduced indefinitely.

1Nelson, M. R., Ghoshal, D., Mejías, J. C., Rubio, D. F., Keith, E., & Roy, K. (2019). A Multi- Niche Microvascularized Human Bone-Marrow-on-a-Chip. bioRxiv, 2019.2012.2015.876813. doi:10.1101/2019.12.15.87681

2Mathur, T., Tronolone, J. J., & Jain, A. (2021). Comparative analysis of blood‐derived endothelial cells for designing next‐generation personalized organ‐on‐chips. Journal of the American Heart Association, 10(22). https://doi.org/10.1161/jaha.121.022795 

3Wan, Z., Zhang, S., Zhong, A. X., Shelton, S. E., Campisi, M., Sundararaman, S. K., Offeddu, G. S., Ko, E., Ibrahim, L., Coughlin, M. F., Liu, T., Bai, J., Barbie, D. A., & Kamm, R. D. (2021). A robust vasculogenic microfluidic model using human immortalized endothelial cells and Thy1 positive fibroblasts. Biomaterials, 276, 121032. https://doi.org/10.1016/j.biomaterials.2021.121032