An Autonomous Tissue Cartridge for Recapitulation of Microvasculature In Vitro
John Morgan, PhD, Jason Spector, M.D..
Weill Medical College of Cornell University, New York, NY, USA.
Tissue engineering seeks to develop physiologically appropriate tissues to restore, maintain or improve function in clinical contexts, and provide platforms with which to study basic biological processes and screen drug candidates and delivery strategies. The complete realization of these goals will represent a paradigm shift. In medicine, it will reduce the use of patient tissue and the associated morbidity at donor sites; in drug development, it will improve the economics and efficacy of early stage drug screening and replace animal testing with appropriate human tissues. We have pioneered approaches to form microvascular networks within 3-D tissue scaffolds. Despite our successes, the overall field has suffered from the lack of a complete suite of tools with which to control these complex cultures with respect to both physical and biological parameters and make them compatible with microsurgery.
Here we present an autonomous tissue cartridge (ATC) for recapitulation of the microvasculature in vitro which solves these problems on three fronts: 1) Precise control of flows within vessels, which can perfuse the microvascular networks with nanoliter-precision control. 2) Hardware that enables fluidic, thermal and gas-phase (O2, CO2, H2O) control of culture parameters in a compact, portable and versatile benchtop platform. Importantly, this platform eliminates the need for conventional incubators and provides live fluorescence imaging during long-term cultures 3) Scaffolding procedures that enable microsurgical anastomosis of an endothelialized vessel within a 3-D matrix in an animal model. Results from the system that study the effect of hemodynamic forces on vascular cells provide new biological insights.
Microvessel tissue templates were constructed in Type I collagen using soft lithography and seeded with human umbilical vein endothelial cells. The fully assembled microfluidic tissue culture device with enclosed microvessels was cultured in the benchtop system with live imaging for 3-7 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). Cell morphology, alignment and migration were analyzed.
The ATC provided consistently stable environmental and temperature control throughout extended cultures. Live imaging revealed dynamic endothelia with cells migrating throughout the vessel walls, both downstream (with) and upstream (against) the flow direction. Cell-cell junctions were contiguous, indicating a confluent, healthy endothelium with intact cytoskeletons. The cross-sectional area of the microvessel expanded and changed profile from the original square cross-section defined lithographically toward an elliptical cross-section with a larger dimension. The displacement of migrating endothelial cells correlated positively with the applied shear stress, with maximum displacement occurring at the highest shear 2.5 Pa (25 dyne/cm2). Similarly, the endothelial cells elongated and aligned in the direction of flow, with net alignment increasing with the magnitude of shear.
This breakthrough sets the stage for clinical translation of the pre-vascularized tissues, provides new insights into relationships between hemodynamic forces and cell morphology/dynamics, and
advances studies of mechano-biology that seek to identify the molecular mechanisms by which endothelial cells sense shear stress.
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