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.

Trends in 3D bioprinting for esophageal tissue repair and reconstruction.

In esophageal pathologies, such as esophageal atresia, cancers, caustic burns, or post-operative stenosis, esophageal replacement is performed by using parts of the gastrointestinal tract to restore nutritional autonomy. However, this surgical procedure most often does not lead to complete functional recovery and is instead associated with many complications resulting in a decrease in the quality of life and survival rate. Esophageal tissue engineering (ETE) aims at repairing the defective esophagus and is considered as a promising therapeutic alternative. Noteworthy progress has recently been made in the ETE research area but strong challenges remain to replicate the structural and functional integrity of the esophagus with the approaches currently being developed. Within this context, 3D bioprinting is emerging as a new technology to facilitate the patterning of both cellular and acellular bioinks into well-organized 3D functional structures. Here, we present a comprehensive overview of the recent advances in tissue engineering for esophageal reconstruction with a specific focus on 3D bioprinting approaches in ETE. Current biofabrication techniques and bioink features are highlighted, and these are discussed in view of the complexity of the native esophagus that the designed substitute needs to replace. Finally, perspectives on recent strategies for fabricating other tubular organ substitutes via 3D bioprinting are discussed briefly for their potential in ETE applications.

Improved visualization and quantitative analysis of drug effects using micropatterned cells.

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck (1-2). To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Contactless electrofunctionalization of a single pore.

Customized pores are smart components that find challenging applications in a variety of fields including purification membranes and biosensing systems. The incorporation of recognition probes within pores is therefore a challenge, due to the technical difficulty of delimiting the area functionalized and obtaining the localized, specific chemical modification of pore walls. An innovative approach, named contactless electrofunctionalization (CLEF), is presented to overcome this problem. CLEF allows easy, one-step modification of the inner surface of a pore etched in a dielectric membrane. The pore wall is coated under the influence of an electric field created by the application of a voltage between two electrodes, located near but not in contact with the pore openings. This specific localization of the deposited material within the pore is extremely rapid. Coatings were reliably and reproducibly obtained using polypyrrole co-polymers bearing oligonucleotides, demonstrating that this technology has a promising future in the design of biosensors. Moreover, the versatility of this process allows the deposition of various electroactive entities such as iridium oxide and therefore indicates a strong potential for diverse applications involving porous materials.

Fluorogenic ester substrates to assess proteolytic activity.

The synthesis of a new type of fluorogenic ester substrates is described. Prepared from fluorescein in three steps with common commercially available precursors, they all generate bright green fluorescence upon proteolysis. Their particular structure allows the same substrate be used to report enzymatic activity of various proteases from serine and cysteine superfamilies. The substrate cleavage is sensitive to specific protease inhibitors providing a tool for inhibitor screening.