Single-Step Biofabrication of In Situ Spheroid-Forming Compartmentalized Hydrogel for Clinical-Sized Cartilage Tissue Formation.

3D cellular spheroids offer more biomimetic microenvironments than conventional 2D cell culture technologies, which has proven value for many tissue engineering applications. Despite beneficiary effects of 3D cell culture, clinical translation of spheroid tissue engineering is challenged by limited scalability of current spheroid formation methods. Although recent adoption of droplet microfluidics can provide a continuous production process, use of oils and surfactants, generally low throughput, and requirement of additional biofabrication steps hinder clinical translation of spheroid culture. Here, the use of clean (e.g., oil-free and surfactant-free), ultra-high throughput (e.g., 8.5 mL min-1 , 10 000 spheroids s-1 ), single-step, in-air microfluidic biofabrication of spheroid forming compartmentalized hydrogels is reported. This novel technique can reliably produce 1D fibers, 2D planes, and 3D volumes compartmentalized hydrogel constructs, which each allows for distinct (an)isotropic orientation of hollow spheroid-forming compartments. Spheroids produced within ink-jet bioprinted compartmentalized hydrogels outperform 2D cell cultures in terms of chondrogenic behavior. Moreover, the cellular spheroids can be harvested from compartmentalized hydrogels and used to build shape-stable centimeter-sized biomaterial-free living tissues in a bottom-up manner. Consequently, it is anticipated that in-air microfluidic production of spheroid-forming compartmentalized hydrogels can advance production and use of cellular spheroids for various biomedical applications.

Mass production of lumenogenic human embryoid bodies and functional cardiospheres using in-air-generated microcapsules.

Organoids are engineered 3D miniature tissues that are defined by their organ-like structures, which drive a fundamental understanding of human development. However, current organoid generation methods are associated with low production throughputs and poor control over size and function including due to organoid merging, which limits their clinical and industrial translation. Here, we present a microfluidic platform for the mass production of lumenogenic embryoid bodies and functional cardiospheres. Specifically, we apply triple-jet in-air microfluidics for the ultra-high-throughput generation of hollow, thin-shelled, hydrogel microcapsules that can act as spheroid-forming bioreactors in a cytocompatible, oil-free, surfactant-free, and size-controlled manner. Uniquely, we show that microcapsules generated by in-air microfluidics provide a lumenogenic microenvironment with near 100% efficient cavitation of spheroids. We demonstrate that upon chemical stimulation, human pluripotent stem cell-derived spheroids undergo cardiomyogenic differentiation, effectively resulting in the mass production of homogeneous and functional cardiospheres that are responsive to external electrical stimulation. These findings drive clinical and industrial adaption of stem cell technology in tissue engineering and drug testing.

Microencapsulated stem cells reduce cartilage damage in a material dependent manner following minimally invasive intra-articular injection in an OA rat model.

Osteoarthritis (OA) is a degenerative disease of the joints for which no curative treatment exists. Intra-articular injection of stem cells is explored as a regenerative approach, but rapid clearance of cells from the injection site limits the therapeutic outcome. Microencapsulation of mesenchymal stem cells (MSCs) can extend the retention time of MSCs, but the outcomes of the few studies currently performed are conflicting. We hypothesize that the composition of the micromaterial's shell plays a deciding factor in the treatment outcome of intra-articular MSC injection. To this end, we microencapsulate MSCs using droplet microfluidic generators in flow-focus mode using various polymers and polymer concentrations. We demonstrate that polymer composition and concentration potently alter the metabolic activity as well as the secretome of MSCs. Moreover, while microencapsulation consistently prolongs the retention time of MSC injected in rat joints, distinct biodistribution within the joint is demonstrated for the various microgel formulations. Furthermore, intra-articular injections of pristine and microencapsulated MSC in OA rat joints show a strong material-dependent effect on the reduction of cartilage degradation and matrix loss. Collectively, this study highlights that micromaterial composition and concentration are key deciding factors for the therapeutic outcome of intra-articular injections of microencapsulated stem cells to treat degenerative joint diseases.

Microfluidic Generation of Thin-Shelled Polyethylene Glycol-Tyramine Microgels for Non-Invasive Delivery of Immunoprotected ß-Cells.

Transplantation of microencapsulated pancreatic cells is emerging as a promising therapy to replenish ß-cell mass lost from auto-immune nature of type I diabetes mellitus (T1DM). This strategy intends to use micrometer-sized microgels to provide immunoprotection to transplanted cells to avoid chronic application of immunosuppression. Clinical application of encapsulation has remained elusive due to often limited production throughputs and body's immunological reactions to implanted materials. This article presents a high-throughput fabrication of monodisperse, non-immunogenic, non-degradable, immunoprotective, semi-permeable, enzymatically-crosslinkable polyethylene glycol-tyramine (PEG-TA) microgels for ß-cell microencapsulation. Monodisperse ß-cell laden microgels of ≈120 µm, with a shell thickness of 20 µm are produced using an outside-in crosslinking strategy. Microencapsulated ß-cells rapidly self-assemble into islet-sized spheroids. Immunoprotection of the microencapsulated is demonstrated by inability of FITC-IgG antibodies to diffuse into cell-laden microgels and NK-cell inability to kill microencapsulated ß-cells. Multiplexed ELISA analysis on live blood immune reactivity confirms limited immunogenicity. Microencapsulated MIN6ß1 spheroids remain glucose responsive for 28 days in vitro, and able to restore normoglycemia 5 days post-implantation in diabetic mice without notable amounts of cell death. In short, PEG-TA microgels effectively protect implanted cells from the host's immune system while being viable and functional, validating this strategy for the treatment of T1DM.

Scalable fabrication, compartmentalization and applications of living microtissues.

Living microtissues are used in a multitude of applications as they more closely resemble native tissue physiology, as compared to 2D cultures. Microtissues are typically composed of a combination of cells and materials in varying combinations, which are dictated by the applications' design requirements. Their applications range wide, from fundamental biological research such as differentiation studies to industrial applications such as cruelty-free meat production. However, their translation to industrial and clinical settings has been hindered due to the lack of scalability of microtissue production techniques. Continuous microfluidic processes provide an opportunity to overcome this limitation as they offer higher throughput production rates as compared to traditional batch techniques, while maintaining reproducible control over microtissue composition and size. In this review, we provide a comprehensive overview of the current approaches to engineer microtissues with a focus on the advantages of, and need for, the use of continuous processes to produce microtissues in large quantities. Finally, an outlook is provided that outlines the required developments to enable large-scale microtissue fabrication using continuous processes.

Scalable Production of Size-Controlled Cholangiocyte and Cholangiocarcinoma Organoids within Liver Extracellular Matrix-Containing Microcapsules.

Advances in biomaterials, particularly in combination with encapsulation strategies, have provided excellent opportunities to increase reproducibility and standardization for cell culture applications. Herein, hybrid microcapsules are produced in a flow-focusing microfluidic droplet generator combined with enzymatic outside-in crosslinking of dextran-tyramine, enriched with human liver extracellular matrix (ECM). The microcapsules provide a physiologically relevant microenvironment for the culture of intrahepatic cholangiocyte organoids (ICO) and patient-derived cholangiocarcinoma organoids (CCAO). Micro-encapsulation allowed for the scalable and size-standardized production of organoids with sustained proliferation for at least 21 days in vitro. Healthy ICO (n = 5) expressed cholangiocyte markers, including KRT7 and KRT19, similar to standard basement membrane extract cultures. The CCAO microcapsules (n = 3) showed retention of stem cell phenotype and expressed LGR5 and PROM1. Furthermore, ITGB1 was upregulated, indicative of increased cell adhesion to ECM in microcapsules. Encapsulated CCAO were amendable to drug screening assays, showing a dose-response response to the clinically relevant anti-cancer drugs gemcitabine and cisplatin. High-throughput drug testing identified both pan-effective drugs as well as patient-specific resistance patterns. The results described herein show the feasibility of this one-step encapsulation approach to create size-standardized organoids for scalable production. The liver extracellular matrix-containing microcapsules can provide a powerful platform to build mini healthy and tumor tissues for potential future transplantation or personalized medicine applications.

On-the-fly exchangeable microfluidic nozzles for facile production of various monodisperse micromaterials.

Microfluidic manufacturing platforms have advanced the production of monodisperse, shape-controlled, and chemically defined micromaterials. However, conventional microfabrication platforms are typically designed and fabricated as single-purpose and single-use tools, which limits their efficiency, versatility, and overall potential. We here present an on-the-fly exchangeable nozzle concept that operates in a transparent, 3D, and reusable microfluidic device produced without cleanroom technology. The facile exchange and repositioning of the nozzles readily enables the production of monodisperse water-in-oil and oil-in-water emulsions, solid and core-shell microspheres, microfibers, and even Janus type micromaterials with controlled diameters ranging from 10 to 1000 µm using a single microfluidic device.