Bioengineered Tubular Biliary Organoids.

Liver organoids have emerged as promising in vitro models for toxicology, drug discovery, and disease modeling. However, conventional 3D epithelial organoid culture systems suffer from significant drawbacks, including limited culture duration, a nonphysiological 3D cystic anatomy with an inaccessible apical surface, and lack of in vivo-like cellular organization. To address these limitations, herein a hydrogel-based organoid-on-a-chip model for the development functional tubular biliary organoids is reported. The resulting constructs demonstrate long-term stability for a minimum duration of 45 d, while retaining their biliary organoid identity and exhibiting key cholangiocyte characteristics including transport activities, formation of primary cilia, and protective glycocalyx. Additionally, tubular organoids are susceptible to physical and chemical injury, which cannot be applied in such resolution to classical organoids. To enhance tissue-level complexity, in vitro formation of a perfusable branching network is induced using a predetermined geometry that faithfully mimics the intricate structure of the intrahepatic biliary tree. Finally, cellular complexity is augmented through co-culturing with vascular endothelial cells and fibroblasts. The models described in this study offer valuable opportunities for investigating biliary morphogenesis and elucidating associated pathophysiological mechanisms.

Addition of High Acyl Gellan Gum to Low Acyl Gellan Gum Enables the Blends 3D Bioprintable.

Long-term stability of gellan gum (GG) at physiological conditions is expected, as very low concentration of divalent ions are required for crosslinking, as compared to alginate­which is extensively used for tissue engineering (TE) applications. Hence, GG is proposed as an ideal candidate to substitute alginate for TE. Deacylated (low acyl; LA) GG forms brittle gels, thus only low concentrations were used for cell encapsulation, whereas acylated (high acyl; HA) GG forms weak/soft gels. 3D bioprinting using pure LAGG or HAGG is not possible owing to their rheological properties. Here, we report development and characterization of bioprintable blends of LAGG and HAGG. Increase in HAGG in the blends improved shear recovery and shape fidelity of printed scaffolds. Low volumetric swelling observed in cell culture conditions over 14 days indicates stability. Volumetric scaffolds were successfully printed and their mechanical properties were determined by uniaxial compressive testing. Mesenchymal stem cells bioprinted in blends of 3% LAGG and 3% HAGG survived the printing process showing >80% viability; a gradual decrease in cell numbers was observed over 21 days of culture. However, exploiting intrinsic advantages of 3D bioprinting, LAGG/HAGG blends open up numerous possibilities to improve and/or tailor various aspects required for TE.

Homeostatic mini-intestines through scaffold-guided organoid morphogenesis.

Epithelial organoids, such as those derived from stem cells of the intestine, have great potential for modelling tissue and disease biology1-4. However, the approaches that are used at present to derive these organoids in three-dimensional matrices5,6 result in stochastically developing tissues with a closed, cystic architecture that restricts lifespan and size, limits experimental manipulation and prohibits homeostasis. Here, by using tissue engineering and the intrinsic self-organization properties of cells, we induce intestinal stem cells to form tube-shaped epithelia with an accessible lumen and a similar spatial arrangement of crypt- and villus-like domains to that in vivo. When connected to an external pumping system, the mini-gut tubes are perfusable; this allows the continuous removal of dead cells to prolong tissue lifespan by several weeks, and also enables the tubes to be colonized with microorganisms for modelling host-microorganism interactions. The mini-intestines include rare, specialized cell types that are seldom found in conventional organoids. They retain key physiological hallmarks of the intestine and have a notable capacity to regenerate. Our concept for extrinsically guiding the self-organization of stem cells into functional organoids-on-a-chip is broadly applicable and will enable the attainment of more physiologically relevant organoid shapes, sizes and functions.

Dimension-Based Design of Melt Electrowritten Scaffolds.

The electrohydrodynamic stabilization of direct-written fluid jets is explored to design and manufacture tissue engineering scaffolds based on their desired fiber dimensions. It is demonstrated that melt electrowriting can fabricate a full spectrum of various fibers with discrete diameters (2-50 µm) using a single nozzle. This change in fiber diameter is digitally controlled by combining the mass flow rate to the nozzle with collector speed variations without changing the applied voltage. The greatest spectrum of fiber diameters was achieved by the simultaneous alteration of those parameters during printing. The highest placement accuracy could be achieved when maintaining the collector speed slightly above the critical translation speed. This permits the fabrication of medical-grade poly(ε-caprolactone) into complex multimodal and multiphasic scaffolds, using a single nozzle in a single print. This ability to control fiber diameter during printing opens new design opportunities for accurate scaffold fabrication for biomedical applications.