Self-Assembly of Prevascular Tissues from Endothelial and Fibroblast Cells Under Scaffold-Free, Nonadherent Conditions

Abstract

To advance the emerging field of bioengineered prevascularized tissues, we investigated factors that control primary vascular network formation in scaffold-free, high-density cell suspension-derived tissues. Fabricating primary vascular networks in a scaffold-free system requires endothelial cells (ECs) and extracellular matrix (ECM)-producing cells that act together to elaborate a permissive matrix. We report findings on the effects to vascular patterning induced by altering the ratio of human endothelial to human fibroblast cells. Analysis revealed that a 1:4 ratio of ECs to fibroblasts resulted in the synthesis of an ECM permissive for organization of primary vascular networks that recapitulated the pattern of primary vascular networks observed in vivo. Importantly this work highlighted the significance of tension in the organization of vascular networks in prevascularized tissues. To our knowledge our in vitro studies are the first to demonstrate the formation of two distinct vascular patterns in an initially homogenous culture system. Specifically, we demonstrate that within our constructs, vascular networks formed with distinct directional orientations that reflect self-assembly-mediated tension. Further, our studies demonstrate that treatment of prevascularized tissues with matrix-promoting factors such as transforming growth factor beta 1 (TGFβ1) increases tissue strength without altering vascular network patterning. Together, the ability to generate prevascularized tissues from human cells in scaffold-free systems and the ability to enhance the strength of the constructs with matrix-promoting factors represent advances to the potential translational utility of prevascularized tissues both as subcutaneous implants and in surgical scenarios requiring the application of tension to the tissue construct.

Implanted Scaffold-Free Prevascularized Constructs Promote Tissue Repair

“…Human endothelial and fibroblast cells at a 1:4 ratio seeded at high density in non-adherent agarose molds formed rod-shaped tissues over the course of three days in culture as previously described 5,6 . Confocal immunofluorescence analysis of whole mount tissues immunolabeled with anti-human CD31 confirmed the presence of organized networks of human endothelial cells (Figure 1).…”

“…Human cell tissue constructs were generated as previously described 6 . Briefly: cell suspension containing human adipose microvascular endothelial cells (ScienCell ™ , Carlsbad, CA) and normal dermal fibroblasts (Lonza, Walkersville, MD) at a 1:4 ratio, respectively, was seeded at high density into non-adherent agarose troughs approximately 0.9cm × 0.1cm × 0.5cm and cultured in 1:2 M200 (Gibco ® , Grand Island, NY) + LSGS (Gibco ® ) + 1% PS (Corning Cellgro ® , Manassas, VA): Hi-glucose DMEM (Gibco ® ) +10% FBS (Corning) + 1% PS for 3 days.…”

“…We have previously identified a method by which prevascular tissues are formed from scaffold-free self-assembly of human endothelial and fibroblast cells 5,6 . During 3 days of in vitro culture, cells self-organized capillary-scale networks of endothelial cells and synthesized an extracellular matrix containing at least laminin, fibronectin, and collagen type I to form prevascular tissue constructs 6 .…”

“…We have previously identified a method by which prevascular tissues are formed from scaffold-free self-assembly of human endothelial and fibroblast cells 5,6 . During 3 days of in vitro culture, cells self-organized capillary-scale networks of endothelial cells and synthesized an extracellular matrix containing at least laminin, fibronectin, and collagen type I to form prevascular tissue constructs 6 . To evaluate the anastomotic potential of these prevascular tissue constructs and their ability to facilitate skeletal muscle repair, tissue constructs were implanted into hind limb muscle defects in athymic mice and immune-competent rats, a model we have used previously to investigate muscle regeneration 1,7,8 .…”

A Modular Strategy to Engineer Complex Tissues and Organs

“…Guiding the tissue engineered construct through the initial phases of vasculogenesis might accelerate neovascularization and anastomosis with host vasculature upon implantation 11. Human umbilical vein endothelial cells (HUVECs) have been cocultured with various stromal supporting cells for vasculogenic purposes 12. Here, HUVECs were cocultured with hMSCs in vasculogenic media to generate prevascular tissue rings, where the cells self‐assembled into early microvascular structures.…”