Growing Vascularized Tissues In Vitro with an Autonomous Tissue Cartridge
John Morgan, Jason Spector.
Cornell University Medical College, New York, NY, USA.
BACKGROUND: Tissue engineering seeks to develop physiologically appropriate tissues to restore, maintain or improve function in clinical contexts, provide platforms to study basic biological processes and screen drug candidates and delivery strategies. It aims to reduce the use of patient tissue and associated morbidity at donor sites; improve economics and efficacy of drug development and screening and replace animal testing with human tissues. Overall, the field has suffered from the lack of a complete toolset to the control physical and biological parameters of these complex cultures and make them compatible with microsurgery. We have pioneered approaches to form microvascular networks within 3-D tissue scaffolds,1-4 and present here an autonomous tissue cartridge (ATC) for growing vascularized tissues in vitro which solves these problems threefold: 1) Precise flow control within vessels, perfusing microvascular networks with nanoliter-precision control. 2) Hardware enabling fluidic, thermal and atmospheric control of cultures in a compact, portable and versatile benchtop platform. Importantly, it eliminates conventional incubators and provides live, multi-wavelength fluorescence imaging 3) Scaffolding that enables microsurgical anastomosis of fully cellularized vessels within 3-D matrices in animal models.
Experimental results from the system are presented showing the effect of hemodynamic forces on vascular cells and the use of adipose-derived stromal and projenitor cells (ADSC) as a cell source for building vascularized tissue constructs.
METHODS: Microvessel tissues were lithographically constructed in collagen and seeded with fluorescently labeled endothelial and perivascular cells, combined with ADSC in the surrounding bulk tissue. The fully assembled microfluidic tissue culture device with enclosed microvessels was cultured in the benchtop system with live imaging for 7-14 days under pump-driven flow using a range of flow rates to achieve physiologic shear stress against the vessel walls , (0.5 Pa, 1.5 Pa, 2.5 Pa). Cellular identity, morphology, alignment and migration were analyzed.
RESULTS: The ATC provided consistently stable environmental and temperature control throughout extended cultures. Live imaging revealed healthy confluent vessels with contiguous cell-cell junctions, intact cytoskeletons, pericyte coverage and dynamic endothelial cells migrating throughout the vessel walls, both downstream (with) and upstream (against) the flow direction. Microvessel cross-sectional areas expanded and changed profile from original lithographically defined squares toward elliptical cross-sections with larger dimensions. ADSC incorporated into the vasculature, influenced by density and proximity. Migrating cell displacement correlated positively with the applied shear stress, with maximum displacement at the highest shear 2.5 Pa. Similarly, cells elongated and aligned in the direction of flow with net alignment increasing with the magnitude of shear.
CONCLUSIONS: This breakthrough sets the stage for future clinical translation of pre-vascularized tissues, provides new insights into relationships between hemodynamic forces, cell morphology and dynamics, and futher advances studies of ADSC fate determination and mechano-biological molecular mechanisms by which cells sense shear stress.
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