Fabrication of 3-dimensional multicellular microvascular structures

Abstract

Despite current advances in engineering blood vessels over 1 mm in diameter and the existing wealth of knowledge regarding capillary bed formation, studies for the development of microvasculature, the connecting bridge between them, have been extremely limited so far. Here, we evaluate the use of 3-dimensional (3D) microfibers fabricated by hydrogel electrospinning as templates for microvascular structure formation. We hypothesize that 3D microfibers improve extracellular matrix (ECM) deposition from vascular cells, enabling the formation of freestanding luminal multicellular microvasculature. Compared to 2-dimensional cultures, we demonstrate with confocal microscopy and RT-PCR that fibrin microfibers induce an increased ECM protein deposition by vascular cells, specifically endothelial colony-forming cells, pericytes, and vascular smooth muscle cells. These ECM proteins comprise different layers of the vascular wall including collagen types I, III, and IV, as well as elastin, fibronectin, and laminin. We further demonstrate the achievement of multicellular microvascular structures with an organized endothelium and a robust multicellular perivascular tunica media. This, along with the increased ECM deposition, allowed for the creation of self-supporting multilayered microvasculature with a distinct circular lumen following fibrin microfiber core removal. This approach presents an advancement toward the development of human microvasculature for basic and translational studies.

Perivascular cells and tissue engineering: Current applications and untapped potential

“…Fibrin microfibers allowed the generation of complex and self-supporting vascular structures including not only the tunica intima made of ECs but also the tunica media composed of SMCs (Barreto-Ortiz et al, 2015). …”

Stem cell-derived vasculature: A potent and multidimensional technology for basic research, disease modeling, and tissue engineering

“…Through these rudimentary vascular models, tube morphogenesis can be studied in the presence of cytokines, defined factors, matrix components, and supporting cells in co-culture [39,41]. These models have become more advanced over the past decade, with more complete control of the 3D topographical cues that guide microvascular development [42,43] and the introduction of genetic controls [44]. These approaches have long been used with primary EC cultures; however, with the increased use of iPSC-derived EC, models of network formation, barrier formation, and sprouting have become more robust and easily modifiable [45].…”

A bench‐top molding method for the production of cell‐laden fibrin micro‐fibers with longitudinal topography

“…Stretching of fibrin hydrogels results in the alignment of the fibrin fibrils (Brown, Litvinov, Discher, Purohit, & Weisel, ). Mixed alginate and fibrin fibers have been produced using an electrospinning technique, with alignment in the hydrogel resulting from a combination of polymer chain alignment as induced by the electrospinning process and mechanical stretching, and endothelial cells seeded on the surface of such fibers adopted aligned patterns of growth that followed the long axis of the fibers (Barreto‐Ortiz et al, , ).…”

“…Here, a new bench‐top method for the fabrication of cell‐laden fibrin fibers with structural anisotropy that guides cell alignment in three‐dimensional (3D) is presented. With respect to extrusion‐based methods including 3D printing, the proposed method is able to produce fibers at low fibrinogen concentration without the use of stabilizing materials such as alginate (Barreto‐Ortiz et al, ; Onoe et al, ; Xu & Wang, ). High fibrinogen concentration enables the production of pure fibrin fibers by extrusion, however with a too dense matrix to allow for cellular activity of the embedded cells, so that the cells are only seeded on the surface of the fibers (Grasman, Pumphrey, Dunphy, Perez‐Rogers, & Pins, ; Jansen, Bacabac, Piechocka, & Koenderink, ).…”