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Engineering Clinically Relevant Hierarchically Vascularized Tissue Flaps Using "Reset"? Vascular Endothelial Cells in Collagen Matrices
Abby Chopoorian Fuchsman
*, Sophia Salingaros, Kate Manley, Riley Mayne, Samuel Medina, Matthew W. Liao, Xue Dong, Jason A. Spector
Weill Cornell Medicine, New York, NY
Background: Our lab has shown that reset vascular endothelial cells (R-VECs) - human umbilical vein endothelial cells transduced to express the angiogenic transcription factor ETV2 - self-assemble into microvascular networks in microfluidic systems using a 3 mg/ml plain collagen matrix. However, higher concentrations of collagen are ideal for tissue engineering due to increased stiffness. This study aims to demonstrate that R-VECs can assemble microvascular networks in a microfluidic system and human-scale "flap" within a collagen matrix.
Methods: For the microfluidic chip, 300,000 R-VECs were suspended in 30 μl of matrix composed of 3, 4, 5, or 6 mg/ml type I collagen. Suspensions were plated in a microfluidic chip and perfused with media by gravitational flow. For the "flap", 10 million GFP-tagged R-VECs were suspended in 2 mL of 8 mg/ml collagen and poured into a mold creating 2 empty channels representing an artery and vein. The construct was perfused with media at 1 dyne using a peristaltic pump. Light and fluorescent microscopy assessed microvascular formation in both models. Average vessel diameter was calculated from 20 random measurements using ImageJ.
Results: Microvascular networks formed after 24 hours in 3, 4, and 5 mg/mL groups. 6 mg/mL collagen was too viscous to flow through the system. Vessels formed in 5 mg/mL were significantly larger than those in 4 and 3 mg/mL (11.36 vs 8.23 [p<0.0001] and 6.53 [p<0.0001] microns) and vessels in 4 mg/mL were significantly larger than those in 3 mg/mL (8.23 vs 6.53[p=0.0409] microns). After 4 days, R-VECs within the 8 mg/ml collagen "flap" showed morphologic and behavioral changes characteristic of early microvascular network formation.
Conclusions: This study demonstrates the formation of microvascular structures by R-VECs within collagen matrices of up to 5 mg/ml in a microfluidic system. R-VECs displayed early microvascular network formation in 8 mg/ml collagen in a 2 cm
3 "flap". Future optimization of this technology holds promise for developing hierarchical vascular networks for tissue engineering.
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