Microvascular engineering for the development of a non-embedded liver sinusoid with a lumen: when endothelial cells do not lose their edge

Abstract

Microvascular engineering seeks to exploit known cell-cell and cell-matrix interactions in the context of vasculogenesis to restore homeostatic or disease development of reliable capillary models in vitro. However, current systems generally focus on recapitulating microvessels embedded in thick gels of extracellular matrix, overlooking the significance of discontinuous capillaries, which play a vital role in tissue-blood exchanges particularly in organs like the liver. In this work, we introduce a novel method to stimulate the spontaneous organization of endothelial cells into non-embedded microvessels. By creating an anisotropic micropattern at the edge of a development-like matrix dome using Marangoni-flow, we achieved a long, non-random orientation of endothelial cells, laying a premise for stable lumenized microvessels. Our findings revealed a distinctive morphogenetic process leading to mature lumenized capillaries, demonstrated with both murine and human immortalized liver sinusoidal endothelial cell lines (LSECs). The progression of cell migration, proliferation and polarization was clearly guided by the pattern, initiating the formation of a multicellular cord that caused a deformation spanning extensive regions and generated a wave-like folding of the gel, hinged at a laminin depleted zone, enveloping the cord with gel proteins. This event marked the onset of lumenogenesis, regulated by the gradual apico-basal polarization of the wrapped cells, leading to the maturation of vessel tight junctions, matrix remodeling, and ultimately the formation of a lumen--recapitulating the development of vessels in vivo. Furthermore, we demonstrate that the process strongly relies on the initial gel edge topography, while the geometry of the vessels can be tuned, from a curved to a straight structure. We believe our facile engineering method, guiding an autonomous self-organization of vessels without the need for supporting cells or complex prefabricated scaffolds, holds promise for future integration into microphysiological systems featuring discontinuous, fenestrated capillaries. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/564881v2_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1d6992org.highwire.dtl.DTLVardef@fe1434org.highwire.dtl.DTLVardef@e07d1aorg.highwire.dtl.DTLVardef@3540ab_HPS_FORMAT_FIGEXP M_FIG C_FIG