Microvascular\nEngineering for the Development of a\nNonembedded Liver Sinusoid with a Lumen: When Endothelial Cells Do\nNot Lose Their Edge

Microvascular engineering seeks to exploit known cell–cell\nand cell–matrix interactions in the context of vasculogenesis\nto restore homeostasis or disease development of reliable capillary\nmodels in vitro. However, current systems generally focus on recapitulating\nmicrovessels embedded in thick gels of extracellular matrix, overlooking\nthe significance of discontinuous capillaries, which play a vital\nrole in tissue-blood exchanges particularly in organs like the liver.\nIn this work, we introduce a novel method to stimulate the spontaneous\norganization of endothelial cells into nonembedded microvessels. By\ncreating an anisotropic micropattern at the edge of a development-like\nmatrix dome using Marangoni flow, we achieved a long, nonrandom orientation\nof endothelial cells, laying a premise for stable lumenized microvessels.\nOur findings revealed a distinctive morphogenetic process leading\nto mature lumenized capillaries, demonstrated with both murine and\nhuman immortalized liver sinusoidal endothelial cell lines (LSECs).\nThe progression of cell migration, proliferation, and polarization\nwas clearly guided by the pattern, initiating the formation of a multicellular\ncord that caused a deformation spanning extensive regions and generated\na wave-like folding of the gel, hinged at a laminin-depleted zone,\nenveloping the cord with gel proteins. This event marked the onset\nof lumenogenesis, regulated by the gradual apico-basal polarization\nof the wrapped cells, leading to the maturation of vessel tight junctions,\nmatrix remodeling, and ultimately the formation of a lumenrecapitulating\nthe development of vessels <i>in vivo</i>. Furthermore,\nwe demonstrate that the process strongly relies on the initial gel\nedge topography, while the geometry of the vessels can be tuned from\na curved to a straight structure. We believe that our facile engineering\nmethod, guiding an autonomous self-organization of vessels without\nthe need for supporting cells or complex prefabricated scaffolds,\nholds promise for future integration into microphysiological systems\nfeaturing discontinuous, fenestrated capillaries.