Self-Organization of Long-Lasting Human Endothelial Capillary-Like Networks Guided by DLP Bioprinting.

Tissue engineering holds great promise for regenerative medicine, drug discovery, and as an alternative to animal models. However, as soon as the dimensions of engineered tissue exceed the diffusion limit of oxygen and nutriments, a necrotic core forms leading to irreversible damage. To overcome this constraint, the establishment of a functional perfusion network is essential. In this work, digital light processing bioprinting is used to encapsulate endothelial progenitor cells (EPCs) in 3D light-cured hydrogel scaffolds to guide them toward vascular network formation. In these scaffolds, EPCs proliferate and self-organize within a few days into branched tubular structures with predefined geometry, forming capillary-like vascular tubes or trees of diameters in the range of 10 to 100 µm. Presenting a confluent monolayer wall of cells strongly connect by tight junctions around a central lumen-like space, these structures can be microinjected with a fluorescent dye and are stable for several weeks in vitro. These endothelial structures can be recovered and manipulated in an alginate patch without altering their shape or viability. This approach opens new opportunities for future applications, such as stacking with other cell sheets or multicellular constructs to yield bioengineered tissue with higher complexity and functionality.

Construction of functional biliary epithelial branched networks with predefined geometry using digital light stereolithography.

Cholangiocytes, biliary epithelial cells, are known to spontaneously self-organize into spherical cysts with a central lumen. In this work, we explore a promising biocompatible stereolithographic approach to encapsulate cholangiocytes into geometrically controlled 3D hydrogel structures to guide them towards the formation of branched tubular networks. We demonstrate that within the appropriate mix of hydrogels, normal rat cholangiocytes can proliferate, migrate, and organize into branched tubular structures with walls consisting of a cell monolayer, transport fluorescent dyes into the luminal space, and show markers of epithelial maturation such as primary cilia and continuous tight junctions. The resulting structures have dimensions typically found in the intralobular and intrahepatic biliary tree and are stable for weeks, without any requirement of bulk supporting material, thereby offering total access to the external side of these biliary epithelial constructs.

Self-Organogenesis from 2D Micropatterns to 3D Biomimetic Biliary Trees.

BACKGROUND AND AIMS: Globally, liver diseases account for 2 million deaths per year. For those with advanced liver disease the only curative approach is liver transplantation. However, less than 10% of those in need get a liver transplant due to limited organ availability. To circumvent this challenge, there has been a great focus in generating a bioengineered liver. Despite its essential role in liver functions, a functional biliary system has not yet been developed. In this framework, exploration of epithelial cell self-organogenesis and microengineering-driven geometrical cell confinement allow to envision the bioengineering of a functional biomimetic intrahepatic biliary tract. APPROACH: three-dimensional (3D) bile ducts were built in vitro by restricting cell adhesion to two-dimensional (2D) patterns to guide cell self-organization. Tree shapes mimicking the configuration of the human biliary system were micropatterned on glass slides, restricting cell attachment to these areas. Different tree geometries and culture conditions were explored to stimulate self-organogenesis of normal rat cholangiocytes (NRCs) used as a biliary cell model, either alone or in co-culture with human umbilical endothelial cells (HUVECs). RESULTS: Pre-seeding the micropatterns with HUVECs promoted luminogenesis with higher efficiency to yield functional branched biliary tubes. Lumen formation, apico-basal polarity, and preservation of the cholangiocyte phenotype were confirmed. Moreover, intact and functional biliary structures were detached from the micropatterns for further manipulation. CONCLUSION: This study presents physiologically relevant 3D biliary duct networks built in vitro from 2D micropatterns. This opens opportunities for investigating bile duct organogenesis, physiopathology, and drug testing.

Rearrangements of the actin cytoskeleton and E-cadherin-based adherens junctions caused by neoplasic transformation change cell-cell interactions.

E-cadherin-mediated cell-cell adhesion, which is essential for the maintenance of the architecture and integrity of epithelial tissues, is often lost during carcinoma progression. To better understand the nature of alterations of cell-cell interactions at the early stages of neoplastic evolution of epithelial cells, we examined the line of nontransformed IAR-2 epithelial cells and their descendants, lines of IAR-6-1 epithelial cells transformed with dimethylnitrosamine and IAR1170 cells transformed with N-RasG12D. IAR-6-1 and IAR1170 cells retained E-cadherin, displayed discoid or polygonal morphology, and formed monolayers similar to IAR-2 monolayer. Fluorescence staining, however, showed that in IAR1170 and IAR-6-1 cells the marginal actin bundle, which is typical of nontransformed IAR-2 cells, disappeared, and the continuous adhesion belt (tangential adherens junctions (AJs)) was replaced by radially oriented E-cadherin-based AJs. Time-lapse imaging of IAR-6-1 cells stably transfected with GFP-E-cadherin revealed that AJs in transformed cells are very dynamic and unstable. The regulation of AJ assembly by Rho family small GTPases was different in nontransformed and in transformed IAR epithelial cells. As our experiments with the ROCK inhibitor Y-27632 and the myosin II inhibitor blebbistatin have shown, the formation and maintenance of radial AJs critically depend on myosin II-mediated contractility. Using the RNAi technique for the depletion of mDia1 and loading cells with N17Rac, we established that mDia1 and Rac are involved in the assembly of tangential AJs in nontransformed epithelial cells but not in radial AJs in transformed cells. Neoplastic transformation changed cell-cell interactions, preventing contact paralysis after the establishment of cell-cell contact and promoting dynamic cell-cell adhesion and motile behavior of cells. It is suggested that the disappearance of the marginal actin bundle and rearrangements of AJs may change the adhesive function of E-cadherin and play an active role in migratory activity of carcinoma cells.