Biofabrication

Emerging efforts to engineer multicellular kidney tissues for drug discovery, disease modeling, and therapeutic repair require scalable generation of kidney organoids derived from human induced pluripotent stem cells (hiPSCs). Here, we report a method for generating kidney ureteric bud and collecting duct epithelial organoids in stirred tank bioreactors. We demonstrate that extracellular matrix microcarriers facilitate organoid expansion and are compatible with suspension bioreactor methods. The combination of cell-coated matrix microcarriers with stirred bioreactors promotes maturation marker expression compared to static controls comprising matrix-embedded organoids. Our method offers promise for generating epithelial organoids that serve as multicellular models and organ building blocks for scalable biofabrication.

The growing demand for three-dimensional (3D) in vitro models that accurately replicate human tissue to investigate disease mechanisms, enable tissue replacement, and support drug testing is driving the need for more advanced manufacturing platforms. Mesenchymal stem/stromal cells (MSCs) are promising candidates for tissue engineering and stem cell therapies due to their regenerative potential and ethical advantages. However, the reproducible and scalable production of 3D models that truly mimic the human complexity and function poses a major challenge. To address these challenges, we have developed a scalable and semi-automated 3D differentiation process for generating osteogenic microtissue derived from MSCs encapsulated in core-shell capsules (CSCs). To validate material-dependent, osteo-inductive effects of hyaluronic acid (HA), we systematically characterised our encapsulation process with regard to CSC integrity, spheroid formation and viability, as well as diffusion and shear thinning properties. Osteogenic differentiation was performed in static and dynamic culture, using either conventional 6-well plate format or a rotating wall vessel bioreactor. HA-CSCs were compared with inert carboxymethyl cellulose (CMC)-CSCs. In addition, hypoxic culture condition and the implementation of human platelet lysate (hPL) further supported physiological relevance. HA led to the formation of larger spheroids with high cell viability and increased metabolic activity compared to CMC. Calcium phosphate deposition within the extracellular matrix (ECM) and elevated ALP activity confirmed successful osteogenesis in all conditions. Furthermore, both HA and dynamic culture promote osteogenesis, and when combined, synergistically enhance the osteogenic differentiation. In conclusion, we present a semi-automated, high-throughput and xeno-free platform process for the 3D differentiation of MSCs towards osteogenic lineage. By integrating HA, hypoxia and dynamic culture conditions, this capsule-based system enhances the physiological relevance, enabling advanced in vitro disease modelling, drug testing, and scalable microtissue generation while reducing reliance on animal models.

Oxygen (O2) transport is a major limitation in high-density cell culture, particularly as three-dimensional (3D) spheroids are increasingly used to better mimic in vivo tissue environments. Here, we developed a poly(4-methyl-1-pentene) (PMP)-based microwell platform, leveraging its lower molecular absorption and greater mechanical stability than the commonly used O2-permeable polydimethylsiloxane (PDMS), while retaining high O2 permeability. We measured O2 permeability in 50-m-thick PMP membranes and found approximately 200-fold greater O2 permeability than the conventional 1-mm polystyrene culture-plate material. PMP membrane-based microwell plates with 350-m-diameter wells maintained spatial separation of pre-formed hepatocyte spheroids while supplying O2 from the plate bottom, synergistically enhancing spheroid oxygenation. Compared with non-O2-permeable plates, the platform reduced localized hypoxia and hypoxia-associated cell injury while improving spheroid viability and growth. Collectively, PMP-based O2-permeable microwell plates provide a promising, scalable, high-throughput platform for spheroid culture with multiple applications in tissue engineering, drug screening and beyond.

Microfluidics-assisted encapsulation of cells in hydrogel microbeads has found diverse applications in tissue engineering, disease modelling, and drug testing. However, upscaled production of submillimeter-sized microbeads, each encapsulating optimally tens to hundreds of living cells, remains a challenge. Excessive viscous stresses or non-homogeneous crosslinking reactions hamper droplet monodispersity, hydrogel integrity, and/or cell viability. Herein, we present a device consisting of (i) tens of parallel step-emulsifiers arranged radially around the hydrogel inlet and (ii) an in-flow UV crosslinking system capable of continuous generation and crosslinking of up to around 45000 microbeads/min, each encapsulating N~50 cells, at cell viability exceeding 80%. We use gelatin methacryloyl (GelMA) as the biomaterial of choice and establish a unique sweet spot in terms of UV wavelength, irradiance, and exposure time- the latter controlled via the net applied flow rate and the length of the UV-exposed microchannel- at which we achieve an optimal balance between long-term microbead stability and cell viability. In particular, we observe a strong correlation between reduced cell viability and poor crosslinking efficiency, the latter attributed to the presence of a spontaneous core-shell structure. We further study the impact of added methacrylated hyaluronic acid (HAMA) and extracellular matrix (ECM) on cell proliferation and microtissue formation in the case of triple-negative breast cancer (TNBC) cells for future application in drug testing. The upscaled production of the encapsulated microtissues demonstrated here provides new perspectives for both drug testing and cell therapies where the precision in microenvironment formulation as well as production throughput remain of primary importance.

Digital micromirror device (DMD)-based bioprinting enables high-resolution fabrication of hydrogel-based tissue constructs, but print fidelity and hydrogel mechanics are typically coupled through polymer concentration, light exposure, and crosslinking density. This creates a materials constraint for vascular bioprinting, where constructs must preserve microscale architecture during fabrication while supporting multicellular remodeling, lumen formation, and long-term tissue organization. To meet these requirements, we developed a 4D GelMA-Cad/gelatin hydrogel with time-dependent mechanics that maintains print fidelity while providing a soft, cell-permissive matrix. GelMA-Cad provides a covalently photocrosslinked, bioactive network containing an N-cadherin-mimetic peptide, while uncrosslinked gelatin serves as a transient matrix-modifying component that diffuses from the hydrogel after fabrication, producing further softening without bulk degradation of the GelMA-Cad network. We characterized hydrogel chemistry with NMR, quantified time-dependent changes in stiffness, visualized pore morphology with cryo-SEM, and evaluated the platform in retinal and pulmonary constructs. The 4D hydrogel supports 3D-patterned accuracy, cell viability, inner blood-retinal barrier vasculature, and vascularized lung-alveolar tissue models. Single-cell RNA-sequencing of the pulmonary vascular-alveolar model demonstrated compartment-specific transcriptional responses, including enrichment of lung capillary-associated endothelial features and AT1-like epithelial states. Overall, this platform decouples DMD print fidelity from post-print matrix mechanics for vascularized soft tissue bioprinting and tissue modeling.

The human pancreas secretes digestive fluid that flows through an extensive ductal network before emptying into the duodenum. However, little is known about how hydrodynamic forces influence pancreatic function and development. We hypothesized that pancreatic cells respond to physiological levels of fluid shear stress. In this study, we developed a microfluidic system featuring reversible sealing between the device layer and the cell culture substrate, enabling (1) conventional open-well cell culture prior to fluid shear stress application and (2) selective patterning of proteins and cells for migration studies. Channel dimensions were chosen to support 24-hour perfusion using standard syringe pumps. Using this platform, we investigated effects of physiological shear stress levels representing basal and activated pancreatic function (2, 8 and 33 mPa) on human iPSC-derived pancreatic progenitor cells. Pancreatic progenitor cells exhibit graded sensitivity to shear stress at the transcriptomic level. Specifically, shear stress upregulated expression of cell cycle-related gene sets; endocrine-associated genes including NEUROG3; and Notch receptor ligands (DLL1, DLL3) in a magnitude-dependent manner. The exposure to unidirectional fluid flow also resulted in a reduction in YAP1 protein content, decreased WWTR1 gene expression, and downregulation of YAP/TAZ target genes. Furthermore, barrier-based migration assays under shear stress revealed that cell patches expanded their boundaries both along and against the flow, with greater distance in the direction of flow. Our system and findings may help lay the foundation for understanding biophysical regulation of developmental processes in a physiologically important, yet hard-to-access, epithelial model system.

Gelatin methacrylate (GelMA) hydrogels offer many advantageous properties such as excellent biocompatibility, tunable stiffness, and rapid fabrication, making them attractive materials for neural systems-on-a-chip devices used to study axonal outgrowth in three-dimensional (3D) environments. In this study, we evaluate properties of GelMA hydrogels crosslinked with visible blue light and application of these hydrogels for encapsulation of primary dorsal root ganglion (DRG) explants inside a custom microscale device. We selected low GelMA polymer concentrations of 3 % and 6 % w/v for hydrogel preparation to create soft materials and demonstrate the effect of degree of functionalization (DoF) on stiffness of GelMA in a blue light-induced crosslinking system. Additionally, a decrease in crosslinking efficiency and stiffness of GelMA was observed upon reconstitution of the polymer in DMEM cell culture media compared to PBS. To evaluate the axonal outgrowth, DRG explants were encapsulated in various GelMA formulations and cultured for 7 days. Following incubation, the explants were fixed and immunochemically labelled with anti-beta-III-tubulin antibody. We further describe an application of custom-developed semi-automated image analysis tool together with linear mixed effects modeling to evaluate axonal outgrowth across different GelMA formulations. Among the formulations tested in our study, 3 % GelMA 95 % DoF hydrogels supported the longest neurite outgrowth suggesting that matrix properties influenced neurite extension.

Granular hydrogels, made of jammed soft microparticles, are of great interest for 3D (bio)printing, as they combine ideal rheological properties and extensive modularity, yielding favorable microstructures for tissue engineering. Typically, the yield-stress properties of these materials, which facilitate printability, are defined by the preparation state and the initial particle content of the hydrogel. Here, we propose a granular hydrogel whose yield-stress and, consequently, printability and printed scaffolds shape retention can be controlled not only by the initial weight fraction of particles but also by their stimuli-responsive swelling. The responsive microgels are prepared from poly(N-isopropylacrylamide) crosslinked by dynamic covalent disulfide bonds. The particles swelling is triggered by reduction-induced cleavage of the disulfide bonds into two pendant thiol groups, enabling printing of multilayer scaffolds with high shape fidelity. Without chemically induced swelling, the same formulation requires higher particle concentrations to be printable. Importantly, the printed structures can be annealed by oxidizing the thiol units back into disulfide inter-particle bonds. The printed scaffolds are compatible with human dermal fibroblasts (HDF) and support cell-material interactions. This system offers a unique opportunity for on-demand modulation of both printability and scaffold microstructure, enabling unique control over local stiffness, packing density, and porosity in granular scaffolds.

Animal-derived surface coatings present the gold standard in stem cell culture. However, they come with major drawbacks such as lot-to-lot inconsistency and an ethically problematic production. Approaches to develop synthetic coating alternatives have largely failed to recapitulate biological functions essential to PSC-self renewal and differentiation. In this study, we have investigated the suitability of a fully synthetic, chemically defined extracellular matrix-mimicking coating named BIOINSTRUCT for hiPSC culture. BIOINSTRUCT was employed during maintenance culture of IMR90-4 cells, as well as directed differentiation of HIHCNi008-A, ACTN2-GFP, and UKKI-011-A cells. BIOINSTRUCT preserved hiPSC characteristics over the experimental period of 10 passages similar to Matrigel. Spontaneous differentiation resulted in the formation of ectodermal, mesodermal and endodermal derivatives, confirming the retention of pluripotency. Directed differentiation on BIOINSTRUCT enabled the generation of neuronal, cardiac and pancreatic endocrine cells. Altogether, our data underscore BIOINSTRUCT as a promising alternative to animal-derived extracellular matrix coatings.

Lack of robustness and high variability in cardiomyocyte (CM) differentiation from human induced pluripotent stem cells (hiPSCs) remain critical challenges for cardiac regenerative medicine and biological research. Guided by developmental principles of lineage specification, we investigated whether early WNT inhibition could expand the process window of CM differentiation and thereby improve robustness. To test this, we compared early WNT inhibition from days 1--5 with standard inhibition from days 3--5 following a 1-day CHIR99021 (CHIR) treatment for WNT activation. Focusing on CHIR concentration as a critical parameter, we found that early WNT inhibition substantially broadened the concentration window yielding [≥]80% differentiation efficiency across distinct hiPSC lines. Extending the analysis to a two-dimensional parameter space incorporating cell density revealed a markedly expanded [≥]80% process window. Temporal analysis of lineage-specific transcription factor expression showed that early WNT inhibition suppressed paraxial mesoderm (PM) specification, likely through MSGN1 suppression, in the range of high initial WNT activation that otherwise favored PM fate, thereby extending the process window. It also suppressed definitive endoderm (DE) specification within the expanded window, promoting a more favorable lateral plate mesoderm/cardiac fate. We further demonstrated substantially reduced variability under parameter perturbations and across wells and batches, accompanied by higher differentiation efficiency, without compromising CM phenotypes. These findings demonstrate early WNT inhibition as an effective strategy for expanding the process window of CM differentiation and improving robustness, while exemplifying the process engineering principle that a broader window can support more robust differentiation, with potential relevance to stem cell--based bioprocesses.

Bone formation and remodeling depend on dynamic biochemical and biomechanical signaling from the collagen-rich osteoid that precedes mineralization, yet the role of osteoid nonlinear mechanics in regulating osteocyte-like network formation remains poorly understood. Here, we engineered a synthetic bottlebrush polymer hydrogel (BB) that mimics key mechanical features of osteoid and compared it to collagen type-I (Col1) matrices with matched shear modulus (~70 Pa) and strain-stiffening behavior. Human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) were cultured for 28 days in growth (GM) or osteogenic media (OM) to examine network formation, and functional connectivity using live cell fluorescence recovery after photobleaching. hMSCs cultured in BB networks and OM showed upregulation of early osteocyte markers compared to Col1. We find that strain-stiffening materials with minimal stress relaxation promote osteocyte-like cell differentiation with functional connectivity, establishing osteoid-mimetic BB hydrogels as a promising matrix to study human osteocytogenesis and osteocyte mechanotransduction in vitro.

In breast cancer, collagen fiber alignment and the second harmonic generation forward-to-backward ratio (F/B), an optical measurement sensitive to fibril-scale organization within collagen fibers, have each been associated with metastatic outcome. Although both are measured at the tumor-stromal interface, in vitro studies have largely examined them separately, because experimental systems have not allowed the two to be varied independently. To address this technical gap, we used a microfluidic biofabrication approach to generate paired aligned and unaligned regions within 3D collagen hydrogels via extensional strain and varied measured F/B across gels by changing the pre-gel pH. The resulting library covered alignment values reported in breast tumors and F/B values associated with poorer metastatic outcome in invasive ductal carcinoma at matched matrix stiffness, pore diameter, and fiber fraction. In MDA-MB-231 cells, alignment biased migration along the fiber axis without changing migration speed, as expected, but the magnitude of the contact guidance response varied with measured F/B. In contrast, MCF-7 cells showed little overall contact guidance, yet migrated ~30% faster in high F/B than low F/B gels in both aligned and unaligned regions. Together, these results show that cells migrated differently in matrices with similar fiber alignment. Thus, including measured F/B may provide additional information to help anticipate how cells migrate in collagen environments that appear similar based on alignment alone.

Type II photoinitiators are essential for numerous applications, including advanced 3D printing techniques such as Xolography. Despite offering tunable wavelengths and reaction mechanisms, their limited photoreactivity hinders widespread adoption. Solutions to increase reactivity, particularly in aqueous solutions and in a biocompatible manner, are urgently needed. Here, we report the surprising finding that iodixanol enhances reactivity of type II photoinitiation systems. Supplementing Xolographic print resins with iodixanol increases photoreactivity, which reduces print times and light dosage tenfold, and enables printing of previously unprintable material formulations. Surprisingly, iodixanol supplementation also improves cell viability in a dose-dependent manner, while reducing cell-mediated light scattering. Together, this enables accurate Xolographic printing of highly cell-dense bioresins, which facilitated the biofabrication of engineered living matter with up to 100 M cells mL-1 (>40% cells by volume) with a resolution of ~65 microns. These advances enabled the first demonstration of tissue function in Xolographically-printed materials. Human induced pluripotent stem cell-derived cardiomyocytes were printed into contractile cardiac-like engineered living matter, which exhibited enhanced cell-cell contact and synchronous contractions, confirming tissue level function. As a highly water soluble, cytocompatible, and clinically approved compound, iodixanol therefore represents a valuable and translatable solution to improve the reactivity of type II photoinitiation systems, which enables volumetric biofabrication of highly cell dense living matter. This unlocks a new role for iodixanol in a wide variety of fields including dentistry, chemistry, organic photovoltaics, tissue engineering, and 3D printing.

A persistent challenge in cartilage tissue engineering is identifying cell sources capable of regenerating cartilage with native-like properties. Variability in chondrogenic potential among human- and animal-derived cells complicates graft development and disease modeling. Here we benchmarked ten chondrogenic cell sources under standardized culture conditions using hyaluronan-alginate biomaterials and implementing a functionality index combining histological and mechanical metrics referenced to native tissue. Mesenchymal stem cells and fetal chondroprogenitors demonstrated superior regenerative capacity, achieving 103% and 95% of native cartilage functionality, respectively. In contrast, adult articular chondrocytes from healthy and osteoarthritic donors produced fibrocartilaginous tissue with functionality scores below 50%. These findings provide a quantitative framework to guide cell source selection and optimize cartilage graft standardization for clinical applications and physiologically relevant disease models, addressing regulatory demands for product characterization.

Developing biomaterials that faithfully recapitulate the complex architecture of native tissues is central to tissue engineering. Conventional hydrogels typically exhibit nanoscale porosity, which limits mass transport and cell migration and thereby constrains tissue development. Here, we exploit liquid-liquid phase separation (LLPS) to generate interconnected macroporous gelatin methacryloyl (GelMA) scaffolds with tunable pore sizes ranging from 2 to 50 m under physiological pH conditions. By mapping the GelMA/dextran phase diagram, we identify a composition range in which LLPS occurs through spinodal decomposition, resulting in a spontaneous bicontinuous, trabecular-like architecture. Pore size can be further tuned by varying polymer concentrations and temperature. We advantageously combined thermal gradients and photopatterning to enable spatial control over pore morphology to fabricate hierarchically structured materials. The biological relevance of these architectures is assessed using fibroblasts cultured within controlled porosity gradients. We show that pore size strongly influences cell infiltration, growth, and spatial organization, with larger pores providing sufficient space for cell migration while reducing the dependence on matrix degradation. Pore size of 7-15 m is identified for maximizing fibroblast growth. Overall, our approach provides a versatile platform for porosity screening, enabling the engineering of biomimetic hydrogels with optimized microarchitectures tailored to specific biological contexts for three-dimensional cell culture.

Influenza A virus (IAV) defective interfering particles (DIPs) are regarded as promising broad-spectrum antivirals due to their high efficacy demonstrated in animal trials. Previously, an OP7 chimera DIP was generated that showed markedly enhanced antiviral efficacy compared to conventional IAV DIPs. In addition, a high-yield cell culture-based production process for OP7 chimera DIPs was established in perfusion mode. However, due to the technical complexity of this technology, we aimed to combine the advantages of perfusion in terms of yield with that of a simple and less complex batch process. In the present work, we established a fed-batch production process for OP7 chimera DIPs that allows a more straightforward implementation into existing production facilities. Using a feeding regime that was able to overcome the cell density effect, we achieved productive virus infection in stirred tank reactors until a cell density of 10E+6 cells/mL. Furthermore, we obtained a high virus titer of 3.8 log10(HAU/100 uL), corresponding to a 3.4-fold increase compared to a reference batch production. Moreover, the produced material demonstrated a significantly increased interfering bioactivity shown by an in vitro potency assay. Interestingly, this efficacy was similarly high as compared to OP7 chimera DIP material previously produced in perfusion mode, despite twofold lower virus titers. Finally, we demonstrate robustness of OP7 chimera DIP seed virus trains, which is significant step towards later pharmaceutical production. Further, corresponding GMP MDCK-PB2(sus) cell and OP7 chimera virus banks are now available for later biomanufacturing further use. In conclusion, the established fed-batch process offers a valid n additional option for pharmaceutical grade OP7 chimera DIP GMP manufacturing production under GMP conditions required for subsequent clinical testing and for later large-scale manufacturing.

Background: Parkinson's disease (PD) is characterized by the progressive loss of midbrain dopaminergic (DA) neurons and aberrant alpha-synuclein (alpha-syn) aggregation, yet disease-modifying therapies that simultaneously retard neurodegeneration and foster regeneration remain lacking. Methods: In this study, we isolated extracellular vesicles (TH and FOXA2 double-positive cell-derived EVs) from TH and FOXA2 double-positive differentiated neural cells, a distinct midbrain floor-plate-derived neural cell population that co-expresses the floor-plate transcription factor FOXA2 together with TH and should therefore not be equated with conventional mature midbrain dopaminergic neurons, and systematically evaluated their therapeutic potential across three complementary models: A53T transgenic mice, 6-OHDA-lesioned rats, and 6-OHDA-treated human midbrain organoids. Integrative transcriptomic and single-cell sequencing analyses, together with toxicological evaluations in rodents and non-human primates, were performed to investigate the underlying mechanisms and safety profile. These TH and FOXA2 double-positive cells were generated using a fully suspension-based, matrix-free, and chemically defined differentiation system devoid of N2/B27 supplements and fetal bovine serum (FBS). This approach offers a simple, controllable, low-cost, and scalable platform with high batch-to-batch consistency for EV manufacturing. Throughout this study, the term "TH and FOXA2 double-positive cells" refers to this distinct TH and FOXA2 double-positive cell population and should not be interpreted as conventional terminally differentiated dopaminergic neurons. Results: Intranasal administration of TH and FOXA2 double-positive cell-derived EVs in A53T mice and 6-OHDA-lesioned rats significantly ameliorated motor deficits, increased nigral tyrosine hydroxylase (TH)-positive neuron counts, reduced alpha-syn aggregation, and suppressed gliosis. In human midbrain organoids, TH and FOXA2 double-positive cell-derived EVs preserved DA neuronal morphology and reduced GFAP and alpha-syn expression. Notably, when EVs were administered simultaneously with 6-OHDA modeling rather than after modeling was completed, the protective effect was stronger. Multi-omics analysis revealed that TH and FOXA2 double-positive cell-derived EVs reversed pathological signatures of interferon-responsive microglia and oxidative stress-adapted astrocytes, while restoring WNT- and non-canonical WNT-mediated intercellular communications. Safety evaluations showed no discernible organ toxicity. Conclusions: TH and FOXA2 double-positive cell-derived EVs exert neuroprotective effects across multiple PD models by modulating specific glial subpopulations and reinstating development-associated signaling pathways. Together with a fully suspension-based, matrix-free, and N2/B27-free differentiation process that enables low-cost, scalable, and highly reproducible manufacturing, TH and FOXA2 double-positive cell-derived EVs represent a promising therapeutic candidate for Parkinson's disease.

Photovoltaic subretinal prostheses replace the lost photoreceptors in patients blinded by atrophic age-related macular degeneration (AMD) and provide central vision by electrically stimulating the inner retinal neurons. Subretinal debris in geographic atrophy separate the inner nuclear layer from the implant by tens of micrometers, increasing the stimulation threshold and reducing the pattern contrast. Absence of this separating layer in animal models diminishes the accuracy of preclinical testing of the next-generation implants, especially with smaller pixels. We developed three-dimensional biomimetic scaffolds nanoprinted onto photovoltaic arrays to provide a precisely controlled separation between the implants and target neurons in animal models of retinal degeneration. Gap sizes of the scaffolds were adjusted to meet the following requirements: they should allow light penetration to the implant, provide unobstructed flow of electric current, but preclude migration of cells into their 3-D structure. Gaps of 3 m were found to be optimal as they restricted cellular migration and maintained the inner nuclear layer above both planar and pillar electrode arrays. Scaffolds did not measurably alter the photovoltaic response and accurately reproduced the distance dependence of stimulation threshold. Following implantation, the initially fluid-filled intra-scaffold space was progressively filled by cellular matter matching the density of the subretinal debris. This transition is expected to increase resistivity of the scaffold to that of tissue, which was evidenced by concurrently improved grating acuity. These results establish a model of the controlled separation between the implant and the target neurons reproducing the features of subretinal debris in geographic atrophy of human patients, thereby providing a platform for accurate preclinical evaluation of the subretinal implants.

Engineered cardiac and skeletal muscle tissues suspended between flexible posts support disease modeling and pharmacology, yet their stimulation, longitudinal imaging and quantitative analysis often remain fragmented and labor-intensive. Here we present Stimulatrix, an open-source platform integrating automated video acquisition within a cell culture incubator, synchronized electrical stimulation and deep learning for longitudinal assessment of contractile tissues in multiwell plates. The platform quantifies tissue compaction, force generation and contraction kinetics with optional cloud processing reducing dependence on local GPU hardware. We demonstrate the workflow in cardiac tissues comprising human induced pluripotent stem cell-derived cardiomyocytes and cardiac fibroblasts, and in primary human skeletal muscle constructs. Force and kinetic measurements were benchmarked against manual annotations. Longitudinal profiling resolved responses to matrix composition and pacing regimens and tracked doxorubicin-associated loss of cardiac contractile force. Stimulatrix provides an accessible workflow for automated functional phenotyping of engineered muscle tissues.

A major challenge in vascular tissue engineering is the development of scalable fabrication methods capable of producing vascular grafts with suitable mechanical properties and hemocompatibility for both preclinical evaluation and future clinical translation. Using a recently developed method to generate acellular vascular grafts from densified collagen hydrogel, we present a proof-of-concept in vivo evaluation of the scalable collagen graft technology. Thick-walled, genipin-crosslinked grafts, representative of a fabrication approach applicable to clinically relevant vessel sizes, were implanted as interposition grafts in the rat abdominal aorta. These grafts supported rapid neointima formation and re-endothelialisation of the luminal surface but developed late thrombosis, highlighting the challenges of evaluating clinically scalable grafts in small-animal models. To enable assessment of the platform in millimetre-scale vessels, we developed a thin-walled (~100 {micro}m), non-crosslinked graft using a constrained densification approach and hyaluronic acid-mediated modulation of collagen fibril assembly to improve compliance matching and hemocompatibility, with the latter providing a matrix-level approach that may complement surface-bound antithrombotic coatings. These grafts remained patent for up to 17 weeks and exhibited neointima formation, re-endothelialisation, and early cellular infiltration into the graft wall. Together, these findings demonstrate in vivo functionality of densified collagen vascular grafts and establish a foundation for future evaluation in larger animal models.