Nitrogen-functionalized graphene quantum dot incorporated GelMA microgels as fluorescent 3D-tissue Constructs.

Biopolymer microgels present many opportunities in biomedicine and tissue engineering. To understand their in vivo behavior in therapeutic interventions, long-term monitoring is critical, which is usually achieved by incorporating fluorescent materials within the hydrogel matrix. Current research is limited due to issues concerning the biocompatibility and instability of the conventional fluorescent species, which also tend to adversely affect the bio-functionality of the hydrogels. Here, we introduce a microfluidic-based approach to generate nitrogen-functionalized graphene quantum dot (NGQD) incorporated gelatin methacryloyl (GelMA) hydrogel microspheres, capable of long-term monitoring while preserving or enhancing the other favorable features of 3D cell encapsulation. A multilayer droplet-based microfluidic device was designed and fabricated to make monodisperse NGQD-loaded GelMA hydrogel microspheres encapsulating skeletal muscle cells (C2C12). Control over the sizes of microspheres could be achieved by tuning the flow rates in the microfluidic device. Skeletal muscle cells encapsulated in these microgels exhibited high cell viability from day 1 (82.9 ± 6.50%) to day 10 (92.1 ± 3.90%). The NGQD-loaded GelMA microgels encapsulating the cells demonstrated higher metabolic activity compared to the GelMA microgels. Presence of sarcomeric α-actin was verified by immunofluorescence staining on day 10. A fluorescence signal was observed from the NGQD-loaded microgels during the entire period of the study. The investigation reveals the advantages of integrating NGQDs in microgels for non-invasive imaging and monitoring of cell-laden microspheres and presents new opportunities for future therapeutic applications.

3D-Printed Tumor-on-Chip for the Culture of Colorectal Cancer Microspheres: Mass Transport Characterization and Anti-Cancer Drug Assays.

Tumor-on-chips have become an effective resource in cancer research. However, their widespread use remains limited due to issues related to their practicality in fabrication and use. To address some of these limitations, we introduce a 3D-printed chip, which is large enough to host ~1 cm3 of tissue and fosters well-mixed conditions in the liquid niche, while still enabling the formation of the concentration profiles that occur in real tissues due to diffusive transport. We compared the mass transport performance in its rhomboidal culture chamber when empty, when filled with GelMA/alginate hydrogel microbeads, or when occupied with a monolithic piece of hydrogel with a central channel, allowing communication between the inlet and outlet. We show that our chip filled with hydrogel microspheres in the culture chamber promotes adequate mixing and enhanced distribution of culture media. In proof-of-concept pharmacological assays, we biofabricated hydrogel microspheres containing embedded Caco2 cells, which developed into microtumors. Microtumors cultured in the device developed throughout the 10-day culture showing >75% of viability. Microtumors subjected to 5-fluorouracil treatment displayed <20% cell survival and lower VEGF-A and E-cadherin expression than untreated controls. Overall, our tumor-on-chip device proved suitable for studying cancer biology and performing drug response assays.

One-Step Bioprinting of Multi-Channel Hydrogel Filaments Using Chaotic Advection: Fabrication of Pre-Vascularized Muscle-Like Tissues.

The biofabrication of living constructs containing hollow channels is critical for manufacturing thick tissues. However, current technologies are limited in their effectiveness in the fabrication of channels with diameters smaller than hundreds of micrometers. It is demonstrated that the co-extrusion of cell-laden hydrogels and sacrificial materials through printheads containing Kenics static mixing elements enables the continuous and one-step fabrication of thin hydrogel filaments (1 mm in diameter) containing dozens of hollow microchannels with widths as small as a single cell. Pre-vascularized skeletal muscle-like filaments are bioprinted by loading murine myoblasts (C2C12 cells) in gelatin methacryloyl - alginate hydrogels and using hydroxyethyl cellulose as a sacrificial material. Higher viability and metabolic activity are observed in filaments with hollow multi-channels than in solid constructs. The presence of hollow channels promotes the expression of Ki67 (a proliferation biomarker), mitigates the expression of hypoxia-inducible factor 1-alpha , and markedly enhances cell alignment (i.e., 82% of muscle myofibrils aligned (in ±10°) to the main direction of the microchannels after seven days of culture). The emergence of sarcomeric α-actin is verified through immunofluorescence and gene expression. Overall, this work presents an effective and practical tool for the fabrication of pre-vascularized engineered tissues.

High-Throughput and Continuous Chaotic Bioprinting of Spatially Controlled Bacterial Microcosms.

Microorganisms do not work alone but instead function as collaborative microsocieties. The spatial distribution of different bacterial strains (micro-biogeography) in a shared volumetric space and their degree of intimacy greatly influences their societal behavior. Current microbiological techniques are commonly focused on the culture of well-mixed bacterial communities and fail to reproduce the micro-biogeography of polybacterial societies. Here, we bioprinted fine-scale bacterial microcosms using chaotic flows induced by a printhead containing a static mixer. This straightforward approach (i.e., continuous chaotic bacterial bioprinting) enables the fabrication of hydrogel constructs with intercalated layers of bacterial strains. These multilayered constructs are used to analyze how the spatial distributions of bacteria affect their social behavior. For example, we show that bacteria within these biological microsystems engage in either cooperation or competition, depending on the degree of shared interface. The extent of inhibition in predator-prey scenarios (i.e., probiotic-pathogen bacteria) increases when bacteria are in greater intimacy. Furthermore, two Escherichia coli strains exhibit competitive behavior in well-mixed microenvironments, whereas stable coexistence prevails for longer times in spatially structured communities. We anticipate that chaotic bioprinting will contribute to the development of a greater complexity of polybacterial microsystems, tissue-microbiota models, and biomanufactured materials.

Using chaotic advection for facile high-throughput fabrication of ordered multilayer micro- and nanostructures: continuous chaotic printing.

This paper introduces the concept of continuous chaotic printing, i.e. the use of chaotic flows for deterministic and continuous extrusion of fibers with internal multilayered micro- or nanostructures. Two free-flowing materials are coextruded through a printhead containing a miniaturized Kenics static mixer (KSM) composed of multiple helicoidal elements. This produces a fiber with a well-defined internal multilayer microarchitecture at high-throughput (>1.0 m min-1). The number of mixing elements and the printhead diameter determine the number and thickness of the internal lamellae, which are generated according to successive bifurcations that yield a vast amount of inter-material surface area (∼102 cm2 cm-3) at high resolution (∼10 µm). This creates structures with extremely high surface area to volume ratio (SAV). Comparison of experimental and computational results demonstrates that continuous chaotic 3D printing is a robust process with predictable output. In an exciting new development, we demonstrate a method for scaling down these microstructures by 3 orders of magnitude, to the nanoscale level (∼150 nm), by feeding the output of a continuous chaotic 3D printhead into an electrospinner. The simplicity and high resolution of continuous chaotic printing strongly supports its potential use in novel applications, including-but not limited to-bioprinting of multi-scale layered biological structures such as bacterial communities, living tissues composed of organized multiple mammalian cell types, and fabrication of smart multi-material and multilayered constructs for biomedical applications.