Modeling Structural Elements and Functional Responses to Lymphatic-Delivered Cues in a Murine Lymph Node on a Chip.

Lymph nodes (LNs) are organs of the immune system, critical for maintenance of homeostasis and initiation of immune responses, yet there are few models that accurately recapitulate LN functions in vitro. To tackle this issue, an engineered murine LN (eLN) has been developed, replicating key cellular components of the mouse LN; incorporating primary murine lymphocytes, fibroblastic reticular cells, and lymphatic endothelial cells. T and B cell compartments are incorporated within the eLN that mimic LN cortex and paracortex architectures. When challenged, the eLN elicits both robust inflammatory responses and antigen-specific immune activation, showing that the system can differentiate between non specific and antigen-specific stimulation and can be monitored in real time. Beyond immune responses, this model also enables interrogation of changes in stromal cells, thus permitting investigations of all LN cellular components in homeostasis and different disease settings, such as cancer. Here, how LN behavior can be influenced by murine melanoma-derived factors is presented. In conclusion, the eLN model presents a promising platform for in vitro study of LN biology that will enhance understanding of stromal and immune responses in the murine LN, and in doing so will enable development of novel therapeutic strategies to improve LN responses in disease.

Biointerface Fiber Technology from Electrospinning to Inflight Printing.

Building two-dimensional (2D) and three-dimensional (3D) micro- and nanofibril structures with designable patterns and functionalities will offer exciting prospects for numerous applications spanning from permeable bioelectronics to tissue engineering scaffolds. This Spotlight on Applications highlights recent technological advances in fiber printing and patterning with functional materials for biointerfacing applications. We first introduce the current state of development of micro- and nanofibers with applications in biology and medical wearables. We then describe our contributions in developing a series of fiber printing techniques that enable the patterning of functional fiber architectures in three dimensions. These fiber printing techniques expand the material library and device designs, which underpin technological capabilities from enabling fundamental studies in cell migration to customizable and ecofriendly fabrication of sensors. Finally, we provide an outlook on the strategic pathways for developing the next-generation bioelectronics and "Fiber-of-Things" (FoT) using nano/micro-fibers as architectural building blocks.

Sustainable electronic textiles towards scalable commercialization.

Textiles represent a fundamental material format that is extensively integrated into our everyday lives. The quest for more versatile and body-compatible wearable electronics has led to the rise of electronic textiles (e-textiles). By enhancing textiles with electronic functionalities, e-textiles define a new frontier of wearable platforms for human augmentation. To realize the transformational impact of wearable e-textiles, materials innovations can pave the way for effective user adoption and the creation of a sustainable circular economy. We propose a repair, recycle, replacement and reduction circular e-textile paradigm. We envisage a systematic design framework embodying material selection and biofabrication concepts that can unify environmental friendliness, market viability, supply-chain resilience and user experience quality. This framework establishes a set of actionable principles for the industrialization and commercialization of future sustainable e-textile products.

Downregulation of extraembryonic tension controls body axis formation in avian embryos.

Embryonic tissues undergoing shape change draw mechanical input from extraembryonic substrates. In avian eggs, the early blastoderm disk is under the tension of the vitelline membrane (VM). Here we report that the chicken VM characteristically downregulates tension and stiffness to facilitate stage-specific embryo morphogenesis. Experimental relaxation of the VM early in development impairs blastoderm expansion, while maintaining VM tension in later stages resists the convergence of the posterior body causing stalled elongation, failure of neural tube closure, and axis rupture. Biochemical and structural analysis shows that VM weakening is associated with the reduction of outer-layer glycoprotein fibers, which is caused by an increasing albumen pH due to CO2 release from the egg. Our results identify a previously unrecognized potential cause of body axis defects through mis-regulation of extraembryonic tissue tension.

Microcapillary cell extrusion deposition with picolitre dispensing resolution.

Extrusion-based cell deposition has become a prominent technique for expanding bioprinting applications. However, the associated print resolution in the order of nanolitre or above has been a limiting factor. The demand for improving print resolution towards the scale of a single cell has driven the development of precision nozzle extrusion, although the benefits gained remain ambiguous. Here, aided by in situ imaging, we investigated the dynamics of cell organisation through an extrusion-based microcapillary tip with picolitre precision through in-air or immersion deposition. The microcapillary extrusion setup, termed 'Picodis', was demonstrated by generating droplets of colouring inks immersed in an immiscible medium. Next, using 3T3 fibroblast cells as an experimental model, we demonstrated the deposition of cell suspension, and pre-aggregated cell pellets. Then, the dynamic organisation of cells within the microcapillary tip was described, along with cell ejection and deposition upon exiting the tip opening. The vision-assisted approach revealed that when dispersed in a culture medium, the movements of cells were distinctive based on the flow profiles and were purely driven by laminar fluid flow within a narrow tip. The primary process limitations were cell sedimentation, aggregation and compaction, along with trapped air bubbles. The use of picolitre-level resolution microcapillary extrusion, although it provides some level of control for a small number of cells, does not necessarily offer a reliable method when a specified number of cells are required. Our study provides insights into the process limitations of high-resolution cell ink extrusion, which may be useful for optimising biofabrication processes of cell-laden constructs for biomedical research. Supplementary information: The online version contains supplementary material available at 10.1007/s42242-022-00205-3.

Deployable extrusion bioprinting of compartmental tumoroids with cancer associated fibroblasts for immune cell interactions.

Realizing the translational impacts of three-dimensional (3D) bioprinting for cancer research necessitates innovation in bioprinting workflows which integrate affordability, user-friendliness, and biological relevance. Herein, we demonstrate 'BioArm', a simple, yet highly effective extrusion bioprinting platform, which can be folded into a carry-on pack, and rapidly deployed between bio-facilities. BioArm enabled the reconstruction of compartmental tumoroids with cancer-associated fibroblasts (CAFs), forming the shell of each tumoroid. The 3D printed core-shell tumoroids showedde novosynthesized extracellular matrices, and enhanced cellular proliferation compared to the tumour alone 3D printed spheroid culture. Further, thein vivophenotypes of CAFs normally lost after conventional 2D co-culture re-emerged in the bioprinted model. Embedding the 3D printed tumoroids in an immune cell-laden collagen matrix permitted tracking of the interaction between immune cells and tumoroids, and subsequent simulated immunotherapy treatments. Our deployable extrusion bioprinting workflow could significantly widen the accessibility of 3D bioprinting for replicating multi-compartmental architectures of tumour microenvironment, and for developing strategies in cancer drug testing in the future.

Constructing biomimetic liver models through biomaterials and vasculature engineering.

The occurrence of various liver diseases can lead to organ failure of the liver, which is one of the leading causes of mortality worldwide. Liver tissue engineering see the potential for replacing liver transplantation and drug toxicity studies facing donor shortages. The basic elements in liver tissue engineering are cells and biomaterials. Both mature hepatocytes and differentiated stem cells can be used as the main source of cells to construct spheroids and organoids, achieving improved cell function. To mimic the extracellular matrix (ECM) environment, biomaterials need to be biocompatible and bioactive, which also help support cell proliferation and differentiation and allow ECM deposition and vascularized structures formation. In addition, advanced manufacturing approaches are required to construct the extracellular microenvironment, and it has been proved that the structured three-dimensional culture system can help to improve the activity of hepatocytes and the characterization of specific proteins. In summary, we review biomaterials for liver tissue engineering, including natural hydrogels and synthetic polymers, and advanced processing techniques for building vascularized microenvironments, including bioassembly, bioprinting and microfluidic methods. We then summarize the application fields including transplant and regeneration, disease models and drug cytotoxicity analysis. In the end, we put the challenges and prospects of vascularized liver tissue engineering.

Cost-Effective Fabrication of Uniformly Aligned Silver Nanowire Microgrid-Based Transparent Electrodes with Higher than 99% Transmittance.

Silver nanowire (Ag NW)-based transparent electrodes (TEs) are promising alternatives to indium tin oxide (ITO) for next-generation flexible optoelectronic devices. Although many different constructs of Ag NW networks and post-treatment methods have been developed for TE applications, trade-offs between optical and electrical performance still remain. Herein, aided by electrohydrodynamic (EHD) printing, we present a cost-effective strategy to fabricate aligned Ag NW microgrids in a large area with excellent uniformity, resulting in superior optoelectronic properties. Guided by the percolation theory and simulation, we demonstrated that by confining aligned Ag NWs into a microgrid arrangement, the percolation threshold can be reduced significantly and adequate electrical conducting pathways can be achieved with an optimized combination of sheet resistance and optical transparency, which surpass conventional random Ag NW networks and random aligned Ag NW networks. The resulting TEs exhibit an ultrahigh transmittance of 99.1% at a sheet resistance of 91 Ω sq-1 with extremely low nanowire usage, an areal mass density of only 8.3 mg m-2, and uniform spatial distribution. Based on this TE design, we demonstrated transparent heaters exhibiting rapid thermal response and superior uniformity in heat generation. Using UV-curable epoxy, highly flexible Ag NW-embedded TEs were fabricated with superior mechanical stabilities and low surface roughness of 2.6 nm. Bendable organic light-emitting diodes (OLEDs) are directly fabricated on these flexible Ag NW electrodes, with higher current efficiency (27.7 cd A-1) than ITO devices (24.8 cd A-1).

A hackable, multi-functional, and modular extrusion 3D printer for soft materials.

Three-dimensional (3D) printing has emerged as a powerful tool for material, food, and life science research and development, where the technology's democratization necessitates the advancement of open-source platforms. Herein, we developed a hackable, multi-functional, and modular extrusion 3D printer for soft materials, nicknamed Printer.HM. Multi-printhead modules are established based on a robotic arm for heterogeneous construct creation, where ink printability can be tuned by accessories such as heating and UV modules. Software associated with Printer.HM were designed to accept geometry inputs including computer-aided design models, coordinates, equations, and pictures, to create prints of distinct characteristics. Printer.HM could further perform versatile operations, such as liquid dispensing, non-planar printing, and pick-and-place of meso-objects. By 'mix-and-match' software and hardware settings, Printer.HM demonstrated printing of pH-responsive soft actuators, plant-based functional hydrogels, and organ macro-anatomical models. Integrating affordability and open design, Printer.HM is envisaged to democratize 3D printing for soft, biological, and sustainable material architectures.

Cancer cell migration on straight, wavy, loop and grid microfibre patterns.

Cell migration plays an important role in physiological and pathological processes where the fibrillar morphology of extracellular matrices (ECM) could regulate the migration dynamics. To mimic the morphological characteristics of fibrillar matrix structures, low-voltage continuous electrospinning was adapted to construct straight, wavy, looped and gridded fibre patterns made of polystyrene (of fibre diameter ca. 3µm). Cells were free to explore their different shapes in response to the directly-adhered fibre, as well as to the neighbouring patterns. For all the patterns studied, analysing cellular migration dynamics of MDA-MB-231 (a highly migratory breast cancer cell line) demonstrated two interesting findings: first, although cells dynamically adjust their shapes and migration trajectories in response to different fibrillar environments, their average step speed is minimally affected by the fibre global pattern; secondly, a switch in behaviour was observed when the pattern features approach the upper limit of the cell body's minor axis, reflecting that cells' ability to divert from an existing fibre track is limited by the size along the cell body's minor axis. It is therefore concluded that the upper limit of cell body's minor axis might act as a guide for the design of microfibre patterns for different purposes of cell migration.

3D printed biomimetic cochleae and machine learning co-modelling provides clinical informatics for cochlear implant patients.

Cochlear implants restore hearing in patients with severe to profound deafness by delivering electrical stimuli inside the cochlea. Understanding stimulus current spread, and how it correlates to patient-dependent factors, is hampered by the poor accessibility of the inner ear and by the lack of clinically-relevant in vitro, in vivo or in silico models. Here, we present 3D printing-neural network co-modelling for interpreting electric field imaging profiles of cochlear implant patients. With tuneable electro-anatomy, the 3D printed cochleae can replicate clinical scenarios of electric field imaging profiles at the off-stimuli positions. The co-modelling framework demonstrated autonomous and robust predictions of patient profiles or cochlear geometry, unfolded the electro-anatomical factors causing current spread, assisted on-demand printing for implant testing, and inferred patients' in vivo cochlear tissue resistivity (estimated mean = 6.6 kΩcm). We anticipate our framework will facilitate physical modelling and digital twin innovations for neuromodulation implants.

On-chip perivascular niche supporting stemness of patient-derived glioma cells in a serum-free, flowable culture.

Glioblastoma multiforme (GBM) is the most common and the most aggressive type of primary brain malignancy. Glioblastoma stem-like cells (GSCs) can migrate in vascular niches within or away from the tumour mass, increasing tumour resistance to treatments and contributing to relapses. To study individual GSC migration and their interactions with the perivasculature of the tumour microenvironment, there is a need to develop a human organotypic in vitro model. Herein, we demonstrated a perivascular niche-on-a-chip, in a serum-free condition with gravity-driven flow, that supported the stemness of patient-derived GSCs and foetal neural stem cells grown in a three-dimensional environment (3D). Endothelial cells from three organ origins, (i) human brain microvascular endothelial cells (hCMEC/D3), (ii) human umbilical vein endothelial cells (HUVECs) and, (iii) human lung microvascular endothelial cells (HMVEC-L) formed rounded microvessels within the extracellular-matrix integrated microfluidic chip. By optimising cell extraction protocols, systematic studies were performed to evaluate the effects of serum-free media, 3D cell cultures, and the application of gravity-driven flow on the characteristics of endothelial cells and their co-culture with GSCs. Our results showed the maintenance of adherent and tight junction markers of hCMEC/D3 in the serum-free culture and that gravity-driven flow was essential to support adequate viability of both the microvessel and the GSCs in co-culture (>80% viability at day 3). Endpoint biological assays showed upregulation of neovascularization-related genes (e.g., angiopoietins, vascular endothelial growth factor receptors) in endothelial cells co-cultured with GSCs in contrast to the neural stem cell reference that showed insignificant changes. The on-chip platform further permitted live-cell imaging of GSC - microvessel interaction, enabling quantitative analysis of GSC polarization and migration. Overall, our comparative genotypic (i.e. qPCR) and phenotypic (i.e. vessel permeability and GSC migration) studies showed that organotypic (brain cancer cells-brain endothelial microvessel) interactions differed from those within non-tissue specific vascular niches of human origin. The development and optimization of this on-chip perivascular niche, in a serum-free flowable culture, could provide the next level of complexity of an in vitro system to study the influence of glioma stem cells on brain endothelium.

Bio-assembling Macro-Scale, Lumenized Airway Tubes of Defined Shape via Multi-Organoid Patterning and Fusion.

Epithelial, stem-cell derived organoids are ideal building blocks for tissue engineering, however, scalable and shape-controlled bio-assembly of epithelial organoids into larger and anatomical structures is yet to be achieved. Here, a robust organoid engineering approach, Multi-Organoid Patterning and Fusion (MOrPF), is presented to assemble individual airway organoids of different sizes into upscaled, scaffold-free airway tubes with predefined shapes. Multi-Organoid Aggregates (MOAs) undergo accelerated fusion in a matrix-depleted, free-floating environment, possess a continuous lumen, and maintain prescribed shapes without an exogenous scaffold interface. MOAs in the floating culture exhibit a well-defined three-stage process of inter-organoid surface integration, luminal material clearance, and lumina connection. The observed shape stability of patterned MOAs is confirmed by theoretical modelling based on organoid morphology and the physical forces involved in organoid fusion. Immunofluorescent characterization shows that fused MOA tubes possess an unstratified epithelium consisting mainly of tracheal basal stem cells. By generating large, shape-controllable organ tubes, MOrPF enables upscaled organoid engineering towards integrated organoid devices and structurally complex organ tubes.

Guided Assembly and Patterning of Intrinsically Fluorescent Amyloid Fibers with Long-Range Order.

Fibrillar amyloids exhibit a fascinating range of mechanical, optical, and electronic properties originating from their characteristic ß-sheet-rich structure. Harnessing these functionalities in practical applications has so far been hampered by a limited ability to control the amyloid self-assembly process at the macroscopic scale. Here, we use core-shell electrospinning with microconfinement to assemble amyloid-hybrid fibers, consisting of densely aggregated fibrillar amyloids stabilized by a polymer shell. Up to centimeter-long hybrid fibers with micrometer diameter can be arranged into aligned and ordered arrays and deposited onto substrates or produced as free-standing networks. Properties that are characteristic of amyloids, including their high elastic moduli and intrinsic fluorescence signature, are retained in the hybrid fiber cores, and we show that they fully persist through the macroscopic fiber patterns. Our findings suggest that microlevel confinement is key for the guided assembly of amyloids from monomeric proteins.

Low-Voltage Continuous Electrospinning: A Versatile Protocol for Patterning Nano- and Micro-Scaled Fibers for Cell Interface.

Nano- and micro-scaled fibers have been incorporated in a number of applications in biofabrication and tissue cultures, providing a cell interfacing structure with extracellular matrix-mimicking topography and adhesion sites, and further supporting localized drug release. Here, we describe the low-voltage electrospinning patterning (LEP) protocol, which allows direct and continuous patterning of sub-micron fibers in a controlled fashion. The processable polymers range from protein (e.g., gelatin) to thermoplastic (e.g., polystyrene) polymers, with flexible selections of collecting substrates. The operation voltage for fiber fabrication can be as low as 50 V, which brings the benefits of reducing costs and mild-processing.

An empirical model to evaluate the effects of environmental humidity on the formation of wrinkled, creased and porous fibre morphology from electrospinning.

Controlling environmental humidity level and thus moisture interaction with an electrospinning solution jet has led to a fascinating range of polymer fibre morphological features; these include surface wrinkles, creases and surface/internal porosity at the individual fibre level. Here, by cross-correlating literature data of far-field electrospinning (FFES), together with our experimental data from near-field electrospinning (NFES), we propose a theoretical model, which can account, phenomenologically, for the onset of fibre microstructures formation from electrospinning solutions made of a hydrophobic polymer dissolved in a water-miscible or polar solvent. This empirical model provides a quantitative evaluation on how the evaporating solvent vapour could prevent or disrupt water vapor condensation onto the electrospinning jet; thus, on the condition where vapor condensation does occur, morphological features will form on the surface, or bulk of the fibre. A wide range of polymer systems, including polystyrene, poly(methyl methacrylate), poly-L-lactic acid, polycaprolactone were tested and validated. Our analysis points to the different operation regimes associated FFES versus NFES, when it comes to the system's sensitivity towards environmental moisture. Our proposed model may further be used to guide the process in creating desirable fibre microstructure.

Inflight fiber printing toward array and 3D optoelectronic and sensing architectures.

Scalability and device integration have been prevailing issues limiting our ability in harnessing the potential of small-diameter conducting fibers. We report inflight fiber printing (iFP), a one-step process that integrates conducting fiber production and fiber-to-circuit connection. Inorganic (silver) or organic {PEDOT:PSS [poly(3,4-ethylenedioxythiophene) polystyrene sulfonate]} fibers with 1- to 3-µm diameters are fabricated, with the fiber arrays exhibiting more than 95% transmittance (350 to 750 nm). The high surface area-to-volume ratio, permissiveness, and transparency of the fiber arrays were exploited to construct sensing and optoelectronic architectures. We show the PEDOT:PSS fibers as a cell-interfaced impedimetric sensor, a three-dimensional (3D) moisture flow sensor, and noncontact, wearable/portable respiratory sensors. The capability to design suspended fibers, networks of homo cross-junctions and hetero cross-junctions, and coupling iFP fibers with 3D-printed parts paves the way to additive manufacturing of fiber-based 3D devices with multilatitude functions and superior spatiotemporal resolution, beyond conventional film-based device architectures.

Broad Bandwidth, Self-Powered Acoustic Sensor Created by Dynamic Near-Field Electrospinning of Suspended, Transparent Piezoelectric Nanofiber Mesh.

Freely suspended nanofibers, such as spider silk, harnessing their small diameter (sub-micrometer) and spanning fiber morphology, behave as a nonresonating acoustic sensor. The associated sensing characteristics, departing from conventional resonant acoustic sensors, could be of tremendous interest for the development of high sensitivity, broadband audible sensors for applications in environmental monitoring, biomedical diagnostics, and internet-of-things. Herein, a low packing density, freely suspended nanofiber mesh with a piezoelectric active polymer is fabricated, demonstrating a self-powered acoustic sensing platform with broad sensitivity bandwidth covering 200-5000 Hz at hearing-safe sound pressure levels. Dynamic near-field electrospinning is developed to fabricate in situ poled poly(vinylidene fluoride-co-trifluoroethylene) (P(VDF-TrFE)) nanofiber mesh (average fiber diameter ≈307 nm), exhibiting visible light transparency greater than 97%. With the ability to span the nanomesh across a suspension distance of 3 mm with minimized fiber stacking (≈18% fiber packing density), individual nanofibers can freely imitate the acoustic-driven fluctuation of airflow in a collective manner, where piezoelectricity is harvested at two-terminal electrodes for direct signal collection. Applications of the nanofiber mesh in music recording with good signal fidelity are demonstrated.

Fabrication of Designable and Suspended Microfibers via Low-Voltage 3D Micropatterning.

Building two-dimensional (2D) and three-dimensional (3D) fibrous structures in the micro- and nanoscale will offer exciting prospects for numerous applications spanning from sensors to energy storage and tissue engineering scaffolds. Electrospinning is a well-suited technique for drawing micro- to nanoscale fibers, but current methods of building electrospun fibers in 3D are restrictive in terms of printed height, design of macroscopic fiber networks, and choice of polymer. Here, we combine low-voltage electrospinning and additive manufacturing as a method to pattern layers of suspended mesofibers. Layers of fibers are suspended between 3D-printed supports in situ in multiple fiber layers and designable orientations. We examine the key working parameters to attain a threshold for fiber suspension, use those behavioral observations to establish a "fiber suspension indicator", and demonstrate its utility through design of intricate suspended fiber architectures. Individual fibers produced by this method approach the micrometer/submicrometer scale, while the overall suspended 3D fiber architecture can span over a centimeter in height. We demonstrate an application of suspended fiber architectures in 3D cell culture, utilizing patterned fiber topography to guide the assembly of suspended high-cellular-density structures. The solution-based fiber suspension patterning process we report offers a unique competence in patterning soft polymers, including extracellular matrix-like materials, in a high resolution and aspect ratio. The platform could thus offer new design and manufacturing capabilities of devices and functional products by incorporating functional fibrous elements.

Solution Formulation and Rheology for Fabricating Extracellular Matrix-Derived Fibers Using Low-Voltage Electrospinning Patterning.

Composite formation and chemical cross-linking are common strategies in tuning the functionality and performance of biologically derived fibers fabricated by electrospinning. The modification to the initial polymeric solution changes the fiber-processing parameters and the associated fiber morphologies. Here, we investigated the gelatin solution formulation and how the addition of homogenized decellularized matrix particles (dCMps) can alter the processability of gelatin fibers produced by low-voltage electrospinning patterning. To produce water-insoluble fibers, the effect of a cross-linker addition was also separately investigated. In particular, we found that the electrospinnability of the solutions formulated with different concentrations of gelatin and dCMps and the morphology of the electrospun fibers were dependent on the rheological properties of the solutions. The solution dispersion rheology can be used as a useful indicator for guiding fiber processability and the fabrication strategy for patterning. The loss tangent associated with an oscillatory rheological test can be used to indicate the switch from an "extrusion-patterning" to a "drag-patterning" configuration. Fine-tuning of the cross-linking time can switch the thin fibrous film between a woven and a nonwoven structure. This study can be used as a guide to producing extracellular matrix fibers and films with specific microstructures suitable for tissue engineering applications.