Growth and Characterization of In Vitro Vascular Tissues

<u>Introduction</u>. Most organs in the body require a perfusable vasculature to deliver needed nutrients, eliminate waste, and maintain normal levels of gas exchange. This is accomplished by means of a hierarchical vascular tree from arteries to capillaries to veins, with the smallest caliber vessels having diameters on the order of 10 mm. To generate realistic in vitro models of healthy and diseased tissue, and for creating implantable organ systems, it is essential to develop the capability to grow microvascular networks (MVN) in our microphysiological systems that recapitulate in vivo morphological complexity and maintain perfusability for as long as needed to characterize and interrogate the model and to prepare systems designed for implantation. Multiple approaches have been tried having the potential to achieve the high cell densities and organ specific microstructure needed to accomplish complex organ functions in a fully-vascularized and perfusable system. Here we describe recent studies aimed at refining the methods employed to optimize the self-organization and self-assembly of a perfusable MVN with long-term viability and functionality.<br/><br/><u>Methods and Results</u>. Our approach has been to focus on the vasculature first, then seek methods to increase organ-specific cell type and density, prolong functionality, and promote anastomosis with intra-organoid vascular structures. We currently use a broad repertoire of endothelial cells (EC) along with a variety of accessory cells, generally selected from the organ of interest. We most often start by suspending the cells in a fibrin gel solution at an optimized concentration (both gel and cells) and allow them to polymerize and self-organize into a 3D network. Polymerized gel moduli are typically in the range of 100-500 Pa initially, which rises to over 1 kPa within 2 weeks as measured by AFM. This stiffening is attributable to secretion of a broad collection of matrix proteins (assessed by mass spectrometry), notably from the collagen family, as well as to the generation of stromal cell-induced stresses (assessed by 3D traction force microscopy).<br/>Methods have been developed to promote robust vascular network formation even when using EC or stromal cell combinations that are otherwise prone to poor vascular development. These methods include the use of interstitial flow (IF) during the initial formation of networks from cells suspended in fibrin gels, and the introduction of intravascular flow after network formation. One of the mechanisms of action with IF is the transient elevation of MMP-2 secretion by the EC during vessel formation, creating conditions that reduce the dependence on stromal cells. Intravascular flow also promotes long-term (&gt; 1 month) viability of the networks, reduced dependency on added growth factors, and a suppression of the initial inflammatory response often observed in in vitro vascular networks.<br/>Recent experiments have also shown that iPS cell-derived EC generated by transient induction of ETV2 expression produce exceptionally high yields of endothelial cells with short-term differentiation protocols that form functional vascular networks in co-culture with various types of stromal cell. Transcriptomic analysis of these endothelial cells indicates strong EC marker expression with few of the epithelial signatures seen in previous iPSC-derived ECs, suggesting wide applicability in numerous model applications.<br/><br/><u>Conclusions</u>. High quality and durable microvascular networks can be reliably formed from a variety of EC/stromal cell combinations for a range of applications. The ECs can be derived from iPSC sources using simple, short-duration protocols and remain viable with good functionality for periods more than one month. These attributes make them excellent candidates for uses in disease models or regenerative medicine applications.