Microvascular engineering seeks to exploit known cell–cell and cell–matrix interactions in the context of vasculogenesis to restore homeostasis 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 nonembedded microvessels. By creating an anisotropic micropattern at the edge of a development-like matrix dome using Marangoni flow, we achieved a long, nonrandom 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 that 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. © 2024 American Chemical Society Subjects what are subjects Biopolymers Cells Chemical structure Extracellular matrix Gelation Keywords what are keywords endothelium self-organization micropatterning folding morphogenesis Read this article To access this article, please review the available access options below. Recommended Access through Your Institution You may have access to this article through your institution. Access Through Université de Paris 6 - Pierre e... Add or Change Institution Purchase Access Read this article for 48 hours. Check out below using your ACS ID or as a guest. Purchase Access Restore my guest access Log in to Access You may have access to this article with your ACS ID if you have previously purchased it or have ACS member benefits. Log in below. Login with ACS ID Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsbiomaterials.4c00939. Additional tests of substrate conditions; additional characterization assays of the substrate during folding; and summary of the identified morphogenetic process (Figures S1–S8) (PDF) Z projection and transversal-view of Stages 3 and 4 (PDF) Phase contrast microscopy movie of TSEC organizing during stage 1 (Movie S1) (AVI) Phase contrast microscopy movie of TSEC organizing during stage 2 (Movie S2) (AVI) Phase contrast microscopy movie of TSEC organizing during stage 3 (Movie S3) (AVI) Phase contrast microscopy movie of TSEC organizing during stage 4 (Movie S4) (AVI) ab4c00939_si_001.pdf (1.18 MB) ab4c00939_si_002.pdf (1.64 MB) ab4c00939_si_003.avi (9.55 MB) ab4c00939_si_004.avi (8.51 MB) ab4c00939_si_005.avi (9.5 MB) ab4c00939_si_006.avi (9.95 MB) Microvascular Engineering for the Development of a Nonembedded Liver Sinusoid with a Lumen: When Endothelial Cells Do Not Lose Their Edge 9 views 0 shares 0 downloads Skip to figshare navigation Supporting informationMicrovascular engineering for the development of a non-embedded liver sinusoid with a lumen: when endothelial cells do not lose their edge.Ana Ximena Monroy-Romero1,2 , Brenda Nieto-Rivera2 , Wenjin Xiao2 , Mathieu Hautefeuille2,*1 Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México, México 2 Laboratoire de Biologie du Développement (UMR 7622), Institut de Biologie Paris Seine, Sorbonne Université, 75005 Paris, France *Corresponding author: Mathieu Hautefeuille. E-mail: mathieu.hautefeuille@sorbonne-universite.frThis PDF file includes:Legends for Movies S1 to S4Supporting textFigures S1 to S8Legends for Movies S1 to S4Movie S1 (separate file). Phase contrast microscopy movie of TSEC organizing during stage 1. Movie S2 (separate file). Phase contrast microscopy movie of TSEC organizing during stage 2.Movie S3 (separate file). Phase contrast microscopy movie of TSEC organizing during stage 3. Movie S4 (separate file). Phase contrast microscopy movie of TSEC organizing during stage 4. Supplementary TextDeciphering optimal substrate conditions for guiding LSEC organization.On a technical note, optimal conditions for the fabrication of GT domes were determined through a series of experiments. We observed that careful handling of the domes is absolutely essential for reproducibility and desired outcome. Experimental outcome was highly sensitive to the initial conditions as discrepancies in cell seeding density, dome contact angle, GelTrex stiffness and culture medium components have led to rapid covering of a monolayer or numerous sprouts appearing at the edge, below the gel; both conditions impeding the morphogenetic steps necessary to form the vessel.For instance, allowing the gel to dry at low humidity or for a longer time in the incubator, and leaving it without culture medium after gelation are unwanted as they all changed the dome properties, increasing stiffening and disturbing the final organization of the cells, preventing cord formation and maturation and promoting the development of a monolayer (Figure S2). On the other hand, manually spreading the droplets resulted in a completely different cell organization outcome (Figure S3). We hypothesized that this was due to the disruption of a cell-size laminin pattern at the beginning of the experiment. The pattern resulted less pronounced at the edge of the structure, with more diffuse and uniform distributions that were larger than a cell size, confirmed by profiles from the immunofluorescence. Interestingly, this led to an interrupted, piecewise tube-like organization, different from that found on intact domes (shown in main text, Figure 3A). This suggests that, similarly to what was found by Nelson's group using collagen I, the organization of non-fibrous, laminin-rich ECM with Marangoni flow also controls adherent cell future patterning. Simple visualization of proposed folding process. The observed mechanism of the folding following lateral strains that leads to the encapsulation of cells by a laminin-rich ECM is not easy to understand intuitively. We kindly suggest the readers to observe what we believe is the closest phenomenon with a simple test. Take a thin piece of soft cloth (e.g. a microfiber cleaning cloth for glasses), grasp it firmly with your two hands between the index and thumb and gently pull it apart. A transverse buckling and folding appears immediately. Intuitively, the greater the distance between the two pulling anchors, the less shear strain required to obtain the buckling (visible as a wrinkling in the piece of soft fabric). This last observation may help explain why there seems to be a minimal cord length needed for the folding process to occur, as regulated by gel edge softness (hence buckling resistance). More experiments employing traction force microscopy with a greater spatiotemporal resolution and nanoindentation to obtain the exact elasticity of the edge will be necessary to try and determine the forces at play here.Summary of the morphogenetic processThe Figure S8 recapitulates the gradual morphogenetic vessel formation described in the manuscript, detailing the stepwise morphogenetic process followed by cells and that was evidenced by our experiments. LSEC spontaneously formed a lumenized capillary-sized vessel. This was achieved by murine and human cells, without external assistance from any mural or stromal cells. The only exogenous support was the presence of an edge of a GelTrex soft matrix, necessary to initiate the whole process which is then completely cell autonomous. This boundary condition was ensured by the Marangoni flow-driven gelation of a GelTrex droplet: it created an ECM density gradient culminating in a thin pattern of concentrated laminin that strictly follows the edge of the dome. This pattern, of one cell-size width, attracted the migration of the endothelial cells from a 50 μm vicinity. Upon adhesion to this ring-like pattern, cells followed this trail: anisotropic migration and proliferation guided a gradual organization of sparse, individual cells into a 1-cell ring-like cord (1D). Upon covering the whole edge, proliferation locally changed to form a multicellular cord (2D) which spread planarly over 2-3 cell widths. Local lateral deformations of the gel provoked a vertical folding process, reminiscent of a surf wave, around a line of hinge cells. The progression of the fold is similar to that of a wave, breaking locally by plunging and extending sideways. Part of the dome advanced towards the gel-glass interface (corresponding shore following the wave analogy). The plunging part curled and wrapped and engulfed the whole multicellular cord, which became an ECM-covered, capillary-sized tube. An apical-basal axis then established, evidenced by podocalyxin localization and the structure entered a lumenogenesis process. It correlated with ZO-1 tight junction maturation and F-actin recruitment in the apical zone and ended up in a microvessel tube with a lumen, running all around the dome. This vessel then slowly matured, as evidenced by the thin BM formation and organization in the direction of the vessel.