Creating tissue-engineered blood vessels as disease models and drug screening platforms

摘要

Introduction: Existing models for drug efficacy and toxicity tests, including human cell lines and animal models, cannot accurately predict how the drugs will behave in people -9 out of 10 experimental drugs fail in clinical studies, even though they pass tests in cell and animal models [FDA news]. Conceivably, in vitro drugs may cause significant functional changes at tissue or organ level but not in conventional 2D cell culture; in vivo, rodent and human response is different. Therefore, physiologically relevant and functional human tissue models have been increasingly valued as alternatives for drug screening. In this work, we report the fabrication of functional tissue-engineered blood vessels (TEBVs) with human cells. Materials and Methods: TEBV fabrication: TEBVs with an inner diameter ＜0.80 mm were fabricated from: ⅰ) sheets of aligned human mesenchymal stem cells (hMSC) (Fig A) and ⅱ) human cells embedded in dense collagen gel. TEBV functional test: Vessel diameters were measured before and after perfusion with vasoconstrictor phenylephrine (1 MM), vasodilators acetylcholine (1 pM), or buffer alone with or without changes in flow rate. Media after perfusion were harvested to measure nitric oxide (NO) release from endothelial cells. Imaging of immune cell adhesion and transendothelium migration: Monocytes and cells to construct TEBVs were labeled with fluorescent dyes. TEBVs were treated with 20ng/mL TNF-a for two hours and then perfused with medium containing HL-60 cells (2x10A5 cell/mL) at 0.5 dynes shear stress for one hour. Confocal images were taken at both the side and the topview of the TEBV lumen to track immune cell perfusion, attachment, and transendothelial migration (TEM) events. 3D images and movies showing cell migration and TEM were reconstructed with Imaris 7.7. Significances indicated as * p＜0.05," p＜0.01 by t-test. Results and Discussion: Confluent aligned hMSC sheets were harvested from nano-grooved PDMS substrate (350nm width, 350nm height, and 700nm periodicity), wrapped circumferentially around a Teflon rod and matured in a rotating wall bioreactor. After further maturation under perfusion for 3 weeks and endothelial progenitor cell coating, vessels increased in strength and exhibited excellent handling and suturing characteristics (Fig A). Flow increased the vessel diameter and nitric oxide (NO) release (Fig B), indicating a functional endothelium. TEBVs exhibited vasoconstriction that increased with the dose of phenylephrine, and vasodilation induced by acetylcholine. To facilitate disease modeling of atherosclerosis, branched TEBVs were fabricated using 3D-printed molds, in which cells were mixed with collagen (1.5mg/mL) to form collagen-gel-TEBV of different sizes (Fig C). Perfusion of monocytes in TEBV with activated endothelium showed that monocytes could attach to the lumen of the TEBV and migrate transendothelially (Fig D, E). With these TEBV models, we were able to monitor different functional changes at tissue level. These models may be applied for drug efficacy and toxicity evaluation as well as disease modeling, like atherosclerosis. Ongoing work includes screening of drugs that have vascular toxicity and fabrication of TEBVs with cells from patients afflicted with genetic disorders. Figure 1. Fabrication of TEBVs and functional tests. A: Schematic diagram of cell-sheet TEBV formation; B: NO release under different perfusion speed and comparison with native vessel; C: Fabrication of branched TEBV using 3D printed molds, made at different sizes (1mm-20mm) with a resolution at 0.1 um; D: Perfusion of monocytes in TEBV; arrows indicate monocytes in perfusion or attached to the lumen of TEBV (Blue: Collagen fibers from second harmonic image; Red: HL-60). E: Left panel is the cross-section view of a TEBV (Blue: cell nucleus; Red: HL-60; Green: cells in TEBV), right panel is the top view of the TEBV lumen (Blue: collagen fiber; Red: cells in TEBV; Green: HL-60). Monocyte adhesion and trans-endothelial migration were observed in real-time. Scale bar in C is 20mm, in D and E are 50μm.