Biofabrication

Embedded extrusion printing can process collagen-rich bioinks, but their low viscosity and slow fibrillogenesis compromise print fidelity and post-deposition stability. Here, we developed a collagen fibril-inducing support bath (FIB) that combines mechanical support for embedded printing with biochemical induction of collagen assembly. Microfibrillated or nanofibrillated cellulose was incorporated into a fibril-inducing buffer, and formulations were screened at 37 degrees Celsius for rheological behaviour and optical transparency. The selected FIB was evaluated by printing 1% and 5% (w/v) articular cartilage-derived extracellular matrix (ECM) inks at 10-20 mm/s and compared with a cellulose-only control bath. FIB exhibited yield-stress, shear-thinning and rapid recovery behaviour that supported reproducible filament deposition. Unlike the control bath, FIB enabled intact construct retrieval following stabilisation and promoted the formation of fibrillar collagen within the printed strands. Scanning electron microscopy revealed D-banded collagen fibrils preferentially oriented along the deposition direction, with dominant orientation peaks within +/- 10-15 degrees. The platform supported the fabrication of 15 x 15 x 1.5 mm sheets and 6 x 6 x 6 mm scaffolds whose macroscopic dimensions were retained after processing. Constructs produced from 5% ECM inks exhibited approximately fourfold higher ramp and relaxation moduli than those produced from 1% ECM inks. Extracts from both formulations caused no detectable reduction in cell metabolic activity after 24 h or 72 h. Mesenchymal stem/stromal cells (MSCs) seeded onto printed sheets became markedly elongated and aligned by day 3, with approximately 80% of cells having an aspect ratio exceeding 1.5, significantly greater than cells seeded onto casted ECM controls, with a mean deviation of ~9 degrees from the filament print direction. These findings establish FIB as a bioactive support bath that couples embedded printability with collagen fibrillogenesis, enabling recoverable collagen-rich constructs with aligned fibrillar architecture that directs early cellular organisation.

Automation of organoid and cell culture processes is essential for achieving scalable and standardized experimentation in regenerative medicine and stem cell research. However, existing microfluidic platforms often rely on complex setups, limiting their integration within standard incubator environments. To address these challenges, we developed a compact, scalable multi-well platform featuring 3D-printed, servo-actuated disposable microvalves for fully automated media and drug exchange. This design eliminates the need for external pressure sources and control channels, providing a simplified and cost-effective solution for organoid culture. The platform integrates an internet-connected microscopy module with a motorized XYZ stage, allowing continuous, real-time imaging of individual wells directly within the incubator. It supports precise and reliable fluid handling under physiological conditions, improving throughput, reproducibility, and accessibility. We validate the platform through bench-top testing and in both mouse and human organoid models. Morphological analysis, immunohistochemistry (IHC), and qPCR demonstrate comparable viability, growth, and gene expression profiles between automated and manual culture conditions. These results establish a robust and scalable framework for fully automated organoid culture, offering a simplified and accessible alternative to conventional microfluidic systems with broad applications in regenerative medicine, drug discovery, and scalable biological screening. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/732526v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@5efd07org.highwire.dtl.DTLVardef@3600d0org.highwire.dtl.DTLVardef@16f85f5org.highwire.dtl.DTLVardef@c39fbd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Three-dimensional (3D) cell culture models, particularly multicellular tumor spheroids, have become essential tools for studying cancer biology, drug screening, and preclinical testing due to their ability to mimic physiological tumor microenvironments. However, traditional invasion assays, such as Boyden-chamber- or Transwell-based systems, often suffer from variability introduced by spheroid handling and transfer, compromising data reproducibility. Here, we present a novel, 3D-printed migration and invasion platform --the MQm-sert-- designed to standardize and streamline spheroid-based invasion assays while maintaining spheroid integrity. Fabricated via fused filament fabrication using biobased polylactic acid, the MQm-sert integrates a hanging-drop spheroid culture system (MQm-sert) with a membrane-based invasion chamber (M-sert), eliminating the need for disruptive spheroid transfer steps. Using synthetic tumor environment mimics (STEMs) composed of breast cancer cells (MDA-MB-231 and MCF7), mesenchymal stromal cells (MSCs), and human pulmonary microvascular endothelial cells (HPMECs), we quantified invasion dynamics and cellular interactions. This innovation significantly reduces experimental variability, as demonstrated by lower variance in invasive cell mass dimensions and cell density compared to conventional workflows. Beyond biological insights, the platform aligns with sustainability goals by leveraging cost-effective, open-source 3D printing, reducing reliance on commercial labware, and addressing key challenges in 3D cell culture standardization.

Conventional extrusion-based 3D bioprinting encounters limitations in fabricating intricate tissue architectures due to fixed nozzle diameters and fixed deposition orientations. These constraints restrict conformal printing on curved or non-planar surfaces and often necessitate support-intensive fabrication strategies. This work introduces a mechanically simplified extrusion platform inspired by the swivel jet nozzle, featuring a free-degree-of-orientation extrusion head termed the universal extrusion head (Univ-Ex head), coupled with a modular nozzle architecture. The Univ-Ex head employs a swivel-like mechanical design that enables orientation freedom without external actuation in its current implementation, thereby minimizing mechanical complexity while supporting deposition on physiologically relevant, non-planar geometries. Multiple nozzle concepts were developed through comparative CAD iterations, with two representative geometries--a flat nozzle and a conical nozzle--selected for experimental validation. The platform is evaluated through parametric CAD design, stereolithography-printed prototypes, proof-of-concept extrusion experiments, and fluid dynamics simulations performed using FLOW-3D software. Numerical and experimental results demonstrate stable filament formation and clear diameter-dependent extrusion behavior, while simulations further confirm the feasibility of angled and non-planar deposition. A variable-diameter nozzle concept is proposed as a forward design direction to enable real-time adjustment of bioink flow rate and deposition resolution in principle; however, the present study intentionally validates the system using fixed-diameter nozzle variants to maintain stable numerical and experimental boundary conditions. A gear-integrated Univ-Ex head is also presented as a forward upgrade and demonstrated as a single-piece prototype. Collectively, this work establishes a scalable, hardware-focused pathway toward conformal bio-additive manufacturing. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/734010v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@8833caorg.highwire.dtl.DTLVardef@33dforg.highwire.dtl.DTLVardef@14d8d11org.highwire.dtl.DTLVardef@685ef0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Modelling human cortical microcircuitry in vitro requires platforms that recapitulate both the compositional complexity and spatial architecture of developing neural tissue. Current organoid and assembloid models often rely on the bulk fusion of pre-differentiated, region-specific cells, lacking the capacity for emergent spatial co-differentiation and microenvironment-driven multiscale organisation. There is also a lack of neural and neuronal-glial models with photo-architectured network geometries. To address these limitations, we present a volumetric in situ differentiation system using a triculture of precision reprogrammed human iPSC-derived glutamatergic neurons, GABAergic neurons and astrocytes embedded throughout ultra-soft photocrosslinkable hydrogel microenvironments. The deterministic and spatially controlled method allows us to engineer macro-scale, interconnected human neural networks directly onto functional microelectrode array interfaces using projection photopatterning for high-throughput screening. Unlike fusion-based organoids and assembloids, our platform enables simultaneous, spatially distributed lineage differentiation and maturation, and extensive topography-guided neurite outgrowth bridging localised cellular hubs to recapitulate various aspects of neurodevelopmental patterning and synaptic integration in 3D. The model enables topographic patterning of neuronal-glial networks as well as 3D cell-embedded bioprinting with the developed triculture system. Both modes of cellular growth are studied and demonstrated here. Longitudinal electrophysiological tracking over a month of culture reveals a transition from immature, quiescent states to asynchronous, information-dense microcircuits characterised by an expanded state-space manifold and physiological excitatory-inhibitory balance. By replicating the mechanics of native brain parenchyma, the model presents a highly reproducible, scalable and flexible platform for the study of cortical microcircuitry development, neurodegenerative decline, and inter-regional network assembly.

Mesenchymal stem/stromal cells (MSCs) are key players in regenerative medicine due to their immunomodulatory properties and ability to promote tissue repair. However, their therapeutic efficacy is often limited by rapid clearance following transplantation. MSC spheroids have shown enhanced functional properties, and we hypothesize that encapsulating them within hydrogel microbeads could offer additional protection and improve their viability. In this study, we developed a novel droplet-based microfluidic protocol for human MSCs derived from the apical papilla (SCAP) encapsulation and In-Bead spheroid formation within alginate microbeads. Optimization of the protocol allowed the formation of MSC spheroids in alginate droplets overnight (In-Bead), before alginate cross-linking and retrieval of alginate beads loaded with MSC spheroids. SCAP were successfully encapsulated within 275 {micro}m alginate microbeads, forming spheroids of approximately 80 {micro}m in diameter. Encapsulated SCAP spheroids retained their immunomodulatory properties. The process was further optimized by incorporating nanomedicines into the alginate solution before the formation of droplets and then spheroids, forming thus hybrid beads (Sph.Beads/NP). Nanomedicines were loaded with NecroX-5, a necrosis inhibitor, to improve SCAP viability further. Live/Dead assays indicated a protective effect of the nanomedicines, supporting the potential of this system for advanced cell delivery in regenerative applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/732109v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@278324org.highwire.dtl.DTLVardef@12bad2org.highwire.dtl.DTLVardef@1a7489forg.highwire.dtl.DTLVardef@190e793_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstractA combination strategy enhancing MSC viability through spheroid formation, microencapsulation, and nanomedicine association achieved by microfluidic encapsulation with In-Bead spheroid formation. Created with BioRender

Three-dimensional (3D) spheroid culture provides a physiologically relevant alternative to conventional two-dimensional culture, but reliable spheroid formation in microwells depends on limiting cell-substrate adhesion. Pluronic F127 is an amphiphilic triblock copolymer that forms a hydrated surface layer, reducing protein adsorption. Here, we evaluated whether this intrinsic anti-fouling property could restore an anti-adhesive surface in used microwell plates to promote spheroid formation. Using chondrogenic ATDC5 and pancreatic {beta}-cell INS-1 cells, we characterized spheroid assembly kinetics, F127 cytotoxicity, surface hydrophilicity, protein adsorption, and spheroid morphology including size and shape factor. Both cell types formed compact spheroids within 24 hours on commercial anti-adhesive microwells. F127 coating markedly reduced water contact angle and protein adsorption, confirming increased surface hydrophilicity and reduced protein fouling. In microwells stripped of their original surface coating, F127 coating amounts of approximately 0.011-0.045 mg/cm2 consistently promoted spheroid formation across both cell types. Soluble F127 concentrations were confirmed to be non-cytotoxic up to 0.625% (w/v), while even complete dissolution of the highest tested coating amount would correspond to only 0.025% (w/v) F127. This simple, reproducible, and low-cost surface-modification strategy may provide an accessible approach for re-functionalizing microwell platforms for 3D cell culture.

We present an active mixing toolhead for extrusion bioprinting. The tool enables the programmable fabrication of tumor microenvironment gradient arrays, through controlled deposition of mixed hydrogel precursor formulations into 384-well plates, pre-seeded with tumor cells. It operates on an open-source bioprinter and can actively mix arbitrary ratios of two hydrogel precursors prior to extrusion. These concentration gradient arrays are compatible with quantitative image analysis of cell viability and morphological responses to hydrogels conditioned with drug or extracellular matrix (ECM) proteins. The tools capacity to mix and print hydrogel precursor gradients was demonstrated using alginate and highly concentrated mCherry-conjugated mini-spidroin solutions. Hydrogel precursor stocks contained fluorescent reporters to facilitate quantifications of mixing efficiency, and as proxies for drug and ECM protein concentrations. The tool was applied to generate hydrogel-based gradients of the apoptosis-inducer staurosporine, from which concentration-dependent MDA-MB-231 breast cancer cell death responses were quantified. Gradient arrays of the ECM protein laminin-511, implicated in breast cancer tumorigenesis, were generated and revealed that increasing laminin-511 concentrations potentiated staurosporine-induced cell death. The study demonstrates the utility of this active mixing toolhead for producing hydrogel gradient arrays, and demonstrates the relevance of studying drug-responses in tumor microenvironment models that account for disease-specific ECM components.

Apical-out intestinal spheroids provide direct access to the lumen-facing epithelial surface, making them attractive three-dimensional models for studying epithelial barrier function, nutrient uptake, and luminal exposure. However, existing polarity-reversal methods typically require releasing spheroids from surrounding ECM gels and culturing them in suspension, which can compromise matrix-derived cues, promote fusion, increase size heterogeneity, and limit scalability. Here, we develop a microfluidic core-shell encapsulation strategy to scalably produce apical-out intestinal spheroids within uniform hydrogel microcapsules. These microcapsules consist of a Matrigel core surrounded by an agarose shell. Flow-focusing microfluidics first confines Caco-2 cells in Matrigel cores that provide instructive extracellular matrix cues, and particle-templated emulsification subsequently encloses each core within an inert agarose shell that prevents spheroid fusion and preserves batch uniformity. The method generated >100,000 microcapsules per experiment, with a mean shell diameter of 117 {micro}m, a coefficient of variation below 9%, and >90% single-spheroid formation efficiency. The resulting spheroids established apical-basolateral polarity, organised tight junctions, formed a dextran-excluding epithelial barrier, and exhibited fatty-acid uptake. This core-shell strategy provides an experimentally tractable platform for scalable intestinal epithelial modelling and may be extensible to other epithelial microtissue systems. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=42 SRC="FIGDIR/small/731294v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@16dc3forg.highwire.dtl.DTLVardef@d5d947org.highwire.dtl.DTLVardef@1abe79forg.highwire.dtl.DTLVardef@f39239_HPS_FORMAT_FIGEXP M_FIG C_FIG

Precise control of growth factor delivery remains a challenge for directing stem cell differentiation in three-dimensional (3D) engineered tissues. In this study, engineered cell-like vesicles are used as programmable microenvironments to enable sustained and localized delivery of growth factors within visible-light-crosslinked GelMA hydrogels. Giant unilamellar vesicles (GUV) loaded with FGF-2 and TGF-{beta}3 were incorporated into bioinks with BM-MSC to drive keratocyte differentiation without repeated soluble growth factor supplementation. ELISA measurements confirmed the removal of non-encapsulated growth factors and the release of the vesicle cargo following induced vesicle rupture. Fluorescence monitoring showed a progressive reduction in detectable FGF-2- and TGF-{beta}3-loaded GUV during culture, while droplet-scale analysis demonstrated the co-deposition of cells and vesicles after printing. After 14 days of differentiation, differentiated cells expressed ALDH1A1, ALDH3A1, lumican, keratocan, and collagen I without induction of -SMA. Interestingly, keratocyte-associated differentiation was retained after drop-on-demand bioprinting, confirmed by qPCR analysis. These findings establish growth factor-loaded vesicles as bioprintable instructive niches capable of supporting localized keratocyte differentiation within 3D corneal constructs.

Synthetic optical skin models offer reproducible, tunable optical properties but lack biological responsiveness, while tissue engineered skin models provide cellular authenticity but suffer from optical variability and limited controllability. The growing demand for alternatives to animal models in the development and validation of optical biomedical technologies highlights the need for a new class of test system that combines the strengths of both approaches while addressing their respective limitations. Here, we introduce the concept of a modular biohybrid skin model, a new testing concept that integrates an optically defined artificial epidermal layer, incorporating polydopamine nanoparticles for changes in skin tone, with living human keratinocytes in two and three-dimensional configurations. In the Optical Protection Model, UV-B-induced apoptosis in primary keratinocytes is quantitatively modulated by model pigmentation level, demonstrating a relationship between optical attenuation and caspase 3/7 activity across three artificial skin tone conditions. In a Structured Dermal Model, keratinocytes seeded onto a hydrogel scaffold localize within follicle-like microcavities, as confirmed by live/dead staining and confocal z-stack imaging. Together, these experiments lead to a new category of test system in the space between inert optical models and variable tissue models that may contribute to reducing the reliance on animal models in biomedical optics.

Engineered epithelial models require three-dimensional extracellular matrix environments that support organized cell growth and allow independent access to luminal and basal compartments. However, many organ-on-chip (OoC) fabrication strategies rely on planar geometries, non-native materials, or multi-step assembly workflows that limit architectural complexity and experimental control. Here, we report a direct in-chip volumetric printing strategy for fabricating stretchable and perfusable collagen-I scaffolds inside custom OoC devices. A vitamin C-regulated ruthenium/sodium persulfate photocrosslinking system enabled high-fidelity printing of collagen-I into open-lumen architectures with ductal- and alveolar-inspired features. By generating scaffolds directly within the final culture device, this workflow eliminates post-print transfer and integrates defined collagen architectures with compartmentalized fluidic access and a mechanically actuable chip format. To support chip-based culture, printed collagen constructs were stabilized after fabrication using EDC/NHS chemistry, which limited thermally induced collagen densification, improved shape retention, and maintained scaffold anchorage during perfusion. The chip design provided separate access to the printed lumen and surrounding basal compartment, which enabled compartment-specific fluid handling while preserving scaffold integrity during inflation, stretching, and perfusion of the printed construct. On the collagen-I scaffolds, human milk-derived mammary epithelial cells formed epithelial layers with tight junctions and lactation associated markers. The platform further supported perfusion culture, in situ staining, and whole-chip volumetric imaging. Together, this work establishes direct in-chip collagen-I volumetric printing as a biofabrication strategy for creating perfusable epithelial tissue chips with native matrix architecture and compartmentalized fluidic control.

Two-photon polymerization (2PP) enables fabrication of hydrogel constructs with submicron, cell-scale resolution, but hydrogel-based bioinks are markedly more sensitive to process variability than conventional photoresists, and this sensitivity is further amplified when living cells are embedded in the resin. Post-processing evaluation, performed only after development, occurs too late to enable any corrective action. A full-factorial design of experiments across laser power and scan speed shows that fabrication outcome depends on both parameter choice and cell presence, with cells shifting and broadening the range of conditions yielding structurally sound constructs. However, substantial variability persists within each nominal condition and cannot be resolved by parameter refinement alone, indicating that outcome is governed by what occurs during each individual print rather than by the parameters set. To capture this, a layer-wise polymerization score is derived from pairwise comparisons of same-layer coaxial images, grounded in the psychophysics of relative judgment, and assembled into a Layer-wise Image Trajectory (LIT) for each print. Applied to both acellular and cell-laden formulations, LIT curves separate cleanly by post-processing outcome without any outcome label used in training, showing that fabrication quality can be predicted early in the build. Building on this signal, individual LIT curves are compared against statistical control limits derived from confirmed successful prints, enabling early detection of anomalous fabrication behavior at early-to-mid layers, well before development. To the best of the authors knowledge, this is the first application of in situ quality prediction and anomaly detection to cell-laden two-photon polymerization.

The increasing prevalence of neurological disorders highlights the need for human in vitro systems that recapitulate key mechanisms of neurodegeneration and neuroinflammation. Although induced pluripotent stem cell (iPSC)-derived organoids and spheroids have advanced structural modelling of the human brain, platforms capable of robustly capturing neuronal electrophysiology in 3D remain limited. Here, we present a neuro-microphysiological system (NeuroMPS) that combines iPSC-derived neurospheres with tailored microelectrode arrays to enable non-invasive, high-resolution monitoring of neuronal network dynamics and functional maturation in vitro. Human iPSC-derived neurospheres, comprising neurons and glial cells, developed synchronous network activity after six weeks of differentiation. The NeuroMPS integrates two key components: a custom microelectrode array with capped electrodes optimized for neurite-level signal detection, and a glass microwell module providing structural confinement and optical compatibility for imaging. This configuration supports stable, longitudinal electrophysiological recordings from three-dimensional neural constructs and enables multimodal analyses. We evaluated platform performance using pharmacological modulators (PTX, TTX, bicuculline, CNQX and 4-AP) and the neurotoxin rotenone through electrophysiological recordings, morphological assessment and metabolic activity profiling. Alterations in network activity were detected within minutes, including at the lowest concentrations tested, whereas corresponding morphological and metabolic changes emerged only at higher doses and later time points. These findings demonstrate the greater sensitivity of electrophysiological readouts in 3D neuronal cultures and their potential for early prediction of compound-induced effects. Collectively, our results establish NeuroMPS as a physiologically relevant, scalable and non-invasive platform for functional interrogation of human iPSC-derived neural networks, with applications in neuropharmacology, neurotoxicology and disease modelling.

Resin three-dimensional (3D) printing is an increasingly popular manufacturing and prototyping method used to create microphysiological systems (MPS), but resin cytotoxicity significantly hinders its adoption, especially when sensitive cell models are incorporated. The mammalian oocyte and early preimplantation embryo consist of cells that are highly sensitive to toxicants and thus represent stringent cell-based models for biocompatibility testing. We developed a Multi-Endpoint Oocyte Safety Assay (MEIOSA) to evaluate the biocompatibility of four ISO 10993 biocompatible BioMed resins (Clear, Durable, Elastic 50A, and Flex 80A). MEIOSA assesses the viability, morphology, meiotic stage, and meiotic spindle morphology of the oocyte after in vitro maturation (IVM). Oocytes were in vitro matured in plate inserts 3D printed with the four BioMed resins. Oocytes cultured in rigid resins (Clear and Durable) or elastomeric resins (Elastic 50A, and Flex 80A) exhibited impaired meiotic progression and complete oocyte degeneration, respectively, relative to controls cultured in polystyrene which matured normally. To determine whether such cytotoxicity could be prevented, we coated the resin inserts with a 5 {micro}m impermeable Parylene-C (PC) barrier. PC coating completely rescued the degeneration and meiotic maturation defect phenotypes for all resins. Remarkably, when the most cytotoxic material (Flex 80A) was coated with PC, the resulting eggs were fertilization-competent and produced embryos capable of normal preimplantation development via in vitro fertilization. Our findings demonstrate that standardized viability-based biocompatibility tests do not identify cytotoxic effects for all cell types and establish MEIOSA as a high sensitivity test model to robustly evaluate biomaterial biocompatibility. Furthermore, PC coating prevents the toxic effects of all resin-3D-printed materials tested, opening up a new toolbox to create MPS compatible with reproductive, and by extension, other sensitive cell cultures. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/730268v1_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@18fab44org.highwire.dtl.DTLVardef@1352e5forg.highwire.dtl.DTLVardef@77889forg.highwire.dtl.DTLVardef@1aabd89_HPS_FORMAT_FIGEXP M_FIG C_FIG

Tympanic membrane perforations remain a common clinical problem, and although surgical intervention through tympanoplasty achieves high success rates, it is associated with donor-site morbidity, surgical complexity and limited restoration of the native radial and circumferential collagen architecture. In this study, 3D extrusion printing was utilized to create an active scaffold and attempt to promote collagen organization through shear-mediated structural alignment. An alginate-carboxymethyl cellulose (CMC) hydrogel with bovine SIS-dECM was prepared and investigated for its suitability as a bioink alternative to tympanoplasty grafts. The physiochemical, rheological and printability characteristics of the hydrogel were assessed. Successful decellularization was confirmed by histological analysis. The incorporation of the SIS-dECM into the hydrogel led to increased swelling, lower apparent viscosity, yield stress and flow stress while maintaining favourable printability and filament stability. Polarized optical microscopy was also used to study the influence of printing speed on the alignment of collagen to mimic the native tympanic membrane radial collagen architecture. Compared with the cast controls, the printed samples presented stronger birefringence signals. Biological evaluation demonstrated that the 15% dECM hydrogel exhibited the highest live cell area percentage and live/dead ratio after 48 h. In addition, the chick chorioallantoic membrane assay demonstrated that the dECM-containing hydrogels improved vascular density. The findings establish a printable, biologically active dECM bioink capable of generating bulk collagen organization through extrusion printing as a platform for tympanic membrane regeneration.

Avascular meniscus tears exhibit minimal intrinsic healing and often progress to joint degeneration due to restricted biological repair capacity and inadequate restoration of tissue-level structure and function. Here, we report a hydrophilic polydopamine (hPDA) fueled bioglue platform that overcomes the solubility limitations of conventional polydopamine (PDA) and enables functional repair of avascular meniscus injuries. Water-soluble hPDA was synthesized via controlled depolymerization and recrystallization, yielding monomeric and oligomeric species rich in catechol, amine, and hydroxyl functionalities. Incorporation of hPDA into fibrin bioglues markedly enhanced mechanical performance, producing 520-525% increases in lap-shear modulus, 165-190% increases in adhesive strength, and a 160% increase in compressive modulus relative to fibrin controls, while degradation was markedly attenuated over 14 days. hPDA exhibited excellent cytocompatibility in both 2D and 3D cultures. In a bovine avascular meniscus explant model, hPDA fueled bioglues promoted tissue integration and aligned collagen remodeling, restoring interfacial mechanics with a 488% increase in tensile modulus and up to 150% higher pull-out strength after 6 weeks. These findings establish hPDA as a versatile bioadhesive building block with strong potential for repairing avascular meniscus tears and other mechanically demanding connective tissues. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/738365v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1f376dborg.highwire.dtl.DTLVardef@10029feorg.highwire.dtl.DTLVardef@1c4f191org.highwire.dtl.DTLVardef@cfa2fe_HPS_FORMAT_FIGEXP M_FIG C_FIG

Tissue engineered skin models are important tools for the in vitro study of physiological and pathophysiological processes as well as the valuation of therapeutic strategies and the efficacy of pharmaceutical and cosmetic compounds. Replicating the functional anatomy of cutaneous tissue is a crucial aspect in ensuring that observations made using these models are translatable to the actual situation in native skin. However, most contemporary full-thickness skin models neglect the reconstruction of the undulated microtopography of the dermal-epidermal junction (DEJ), which not only contributes to the biological functionality of the skin (e.g. stem cell niches), but also affects tissue mechanics and drug diffusion. Herein, we fabricated bilayer skin models with DEJ-like microtopographies introduced by interfacial wrinkling between a hydrogel and a nanofibrous membrane through a controllable swelling-deswelling approach. The interfacial wrinkles contributed to the structural integrity of the bilayer models. Their formation could be induced in the presence of living cells through mechanical stress-driven buckling instabilities, thus differentiating the process from commonly used pre-patterning techniques. Bilayer models supported the co-culture of human dermal fibroblasts and human epidermal keratinocytes, and the formation of stratified epithelia. Our findings provide a potential alternative method to introduce DEJ-like anatomical features into full-thickness skin tissue models.

Organoids-on-chip combine the 3D complex microenvironment and cellular composition of organoids with microfluidic flow, increasing nutrient-waste exchange and mimicking the contributions of in vivo interstitial and vascular flow. However, the widespread adoption of organoid-on-chip platforms is limited by the lack of incubator-friendly flow control systems. Existing approaches often rely on commercially available syringe or peristaltic pumps, but these are bulky, lack scalability, and present a significant barrier for clinical translation. To circumvent these issues, we present the Compact Active Perfusion Standalone Organoid-on-Chip (CAPS-OC) platform, a fully integrated and battery-powered microfluidic system capable of culturing organoids in active media flow. To achieve this, we introduce a novel low-power mechanism of pressure pulse generation using an off-the-shelf compact rotary actuator (CRA), and package it into a compact electromechanical assembly. This assembly provides timed pneumatic inputs to achieve programmable control of membrane-based peristaltic pumps, with [~]100 {micro}L/hr dynamic range achieved on a custom microfluidic organoid chip. We biologically validated this system by culturing pancreatic cancer organoids derived from a KrasLSL-G12D/+; Trp53LSL-R172H/WT; Pdx1-Cre (KPC) genetically engineered mouse model. We found that our chip enhances proliferation and helps sustain a population of larger (>150 {micro}m) organoids compared to standard dome based static culture. Additionally, through immunostaining, we observe that KPC organoids cultured in the chip show more aggressive PDAC phenotype with reduced nuclear expression of GATA6, whereas organoids in static culture retain less aggressive classical- like subtype. Finally, testing of a RAS inhibitor drug, daraxonrasib, on the KPC organoids on chip showed size-based sensitivity elucidating the impact of active perfusion on the drug diffusion kinetics. Altogether, we establish CAPS-OC as a valuable tool for the bioengineering community and for clinically translating organoid model systems.

The environmental impact of synthetic textiles has prompted the search for sustainable and biodegradable alternatives. This study correlates the growth conditions used to produce kombucha-derived cellulose non-woven mats with their mechanical performance as a function of post-processing. Systematically, the fermentation and growth parameters of the non-wovens, including inoculum density, carbon-source loading, temperature, and pH value were investigated. Thick, uniform non-wovens were obtained using mildly acidic conditions that balanced nutrient availability and growth rate, moderate inoculum and carbon loading at 30 {degrees}C. Next, we used uniaxial tensile testing and rheology to thoroughly compare the mechanical properties of two post-processing routes, lyophilization and oven-drying against the as-produced wet non-wovens. Overall, the lyophilized non-wovens displayed the highest ultimate tensile strength (14.36 {+/-}0.9 MPa) and elongation at break (24.54 {+/-}1.9%), which were statistically greater than the oven-dried (2.54 {+/-}0.3 MPa, 6.03 {+/-}0.8%) and the wet non-wovens (1.66 {+/-}0.3 MPa, 9.35 {+/-}2.8%). We conclude by performing a proof-of-concept recyclability experiment: we showed that kombucha-derived clothing could be enzymatically degraded and then re-manufactured into new nanofibers by electrospinning. Together, these results demonstrate a circular pathway encompassing the growth and processing of mechanically robust kombucha-derived cellulose non-wovens, as well as their biodegradation and re-manufacturing.