Bioengineering & Translational Medicine

Snake venom composition and its contribution to toxic effects has been heavily researched, but there is a comparative lack of information on venoms fluidic properties and their relationship with fang morphology during the envenomation process. Understanding how venom flows through a fang can shed light on bite site dynamics and potentially explain bite symptoms. In this article we first conduct a broad comparative test of the rheological properties of venom from thirteen snake species, including multiple viperid and elapid snake species, revealing a shear-thinning non-Newtonian flow behavior in all studied species. However, we have not observed strong phylogenetic signal in venom fluidic properties, suggesting that flow properties may vary independently of evolutionary relationships between snake species. Second, we demonstrate that snake venoms fluidic properties can be modeled by other inexpensive, safe, and abundant shear-thinning surrogate fluids. We found that aqueous solutions of bovine serum albumin protein and xanthan gum are useful venom mimics, matching the rheological behavior of venoms from the studied snake species across a range of relevant shear rates. We further evaluated the performance of these snake venom mimics in a simulated venom delivery system, showing good and robust mimetic control of the flow properties as a function of applied pressure. By elucidating the fluidic properties of snake venom and providing a non-toxic, scalable surrogate fluid model to be used in further studies, we provide the biomedical, toxicology, evolutionary biology communities with a tool to study envenomation physics in an inexpensive and safe fashion. We suggest it is possible to design species-specific venom mimics that facilitate research on the biomechanics and fluid dynamics of venom delivery via snake bites, and inform the design of bioinspired puncture and injection devices.

Intracranial aneurysm (IA) rupture is catastrophic, yet current models of rupture-risk inadequately capture underlying IA remodelling mechanisms. Endothelial-haemodynamic interactions are central to these processes, but in vitro flow platforms often lack vessel-relevant geometry or long-term perfusion. Here, temporal and spatial endothelial responses to haemodynamic stress were investigated across idealised and patient-specific vascular models. Polydimethylsiloxane models were endothelialised with human aortic endothelial cells then perfused at up to 1.6 Pa wall shear stress for five days. IA models were exposed to steady or cardiovascular flow waveforms, with endothelial phenotype assessed by immunofluorescence and cytokine profiling. Flow initiation induced a transient inflammatory response, with elevated MCP-1 and TNF- at day two, followed by a resolution of cytokine levels by day five, including a [~]7.5-fold reduction in MCP-1, despite increased haemodynamic loading. Endothelial cells retained a cobblestone-like morphology with eNOS undetected, resembling a partially activated phenotype. Compared with steady flow, cardiovascular flow reduced TGF-{beta}1 and IL-8 secretion and decreased FGF-b consumption ([~]2.5 fold), suggesting enhanced phenotypic stability. This study presents the first in vitro IA model incorporating a cardiovascular flow waveform and identifies cytokine signatures with potential utility as biomarkers of IA remodelling, highlighting the importance of long-term perfusion for modelling chronic vascular disease. Table of Contents FigureAn in vitro model of an intracranial aneurysm was developed to investigate how fluid flow dynamics impact endothelial remodelling and inflammation. Pulsatile cardiac flow promoted stabilisation of inflammatory signalling, which was sustained under a steady flow regime. Cytokine signatures emerged with potential utility as biomarkers of IA remodelling, highlighting the importance of long-term perfusion for modelling chronic vascular disease. The schematic of the cytokine release dynamics used in the graphical abstract below was generated with the assistance of AI-based tools including ChatGPT (v5.5) and M365 Copilot to align with key results from this manuscript. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/733289v1_ufig1.gif" ALT="Figure 1000"> View larger version (80K): org.highwire.dtl.DTLVardef@c00d12org.highwire.dtl.DTLVardef@9a3afaorg.highwire.dtl.DTLVardef@1961b66org.highwire.dtl.DTLVardef@1e0fec5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Oxidative stress-induced airway injury contributes to chronic obstructive pulmonary disease (COPD). Cardiovascular complications increase COPD morbidity and mortality, but mechanistic links between airway injury and vascular dysfunction remain unclear, largely due to limitations of in vitro models that fail to replicate the multicellular lung environment. We developed REVAS, a modular organ-on-chip platform to study human respiratory-vascular cell-cell interactions at baseline and under oxidative stress conditions. REVAS consists of two respiratory chips hosting airway epithelium and microvascular endothelium, and a vascular chip hosting pulmonary artery endothelial cells co-cultured with vascular support cells, including smooth muscle cells, pericytes and fibroblasts. We studied effects of vascular support and respiratory cells on vascular endothelial phenotype at baseline and under H2O2-induced epithelial oxidative stress using functional assays, proteomic and transcriptomic analyses. Multicellular environment enhanced vascular endothelial barrier function and promoted respiratory and vascular cell differentiation at baseline. Mural cells altered endothelial cell-matrix interactions, metabolism and cytoskeletal remodelling, while respiratory cells promoted endothelial aerobic respiration and quiescent phenotype. Epithelial oxidative stress triggered inflammatory gene expression across all respiratory and vascular cells alongside apoptotic, reparative and pro-angiogenic signalling in endothelial and mural cells, accompanied by increased release of COPD-relevant cytokines and chemokines, including IL-6, TNF-/{beta}, IL-8, CCL5, CXCL9, PDGF, TGF-{beta}. Comparative analyses with COPD endothelial datasets confirmed that REVAS recapitulates key features of disease-associated endothelial dysfunction. These findings demonstrate that airway epithelial injury drives downstream vascular responses linked to inflammation and vascular remodelling, establishing REVAS as a human-relevant platform for mechanistic and therapeutic evaluation of cell-cell interactions in COPD and related lung diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/730087v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@e29fd8org.highwire.dtl.DTLVardef@6c3d22org.highwire.dtl.DTLVardef@21a53forg.highwire.dtl.DTLVardef@e7f432_HPS_FORMAT_FIGEXP M_FIG C_FIG REVAS: a microfluidic platform developed to model multicellular interactions between airway epithelium and pulmonary vasculature under basal and oxidative stress. COPD: Chronic Obstructive Pulmonary Disease; EMT: endothelial-to-mesenchymal transition; HsEpCs: human small airway epithelial cells; HPMVECs: human pulmonary microvascular endothelial cells; HPAECs: human pulmonary artery endothelial cells; HPASMCs: human pulmonary artery smooth mucle cells; HPFs: human pulmonary fibcroblasts; HPCs: human pericytes.

ObjectivesTo determine how selective optogenetic vagus nerve stimulation (VNS) of distinct axonal subpopulations modulates systemic inflammatory cytokines in an acute model of endotoxemia. Materials and MethodsA silicone spiral nerve cuff with integrated custom probes including microscale light-emitting diodes (LEDs) was fabricated and implanted on the left cervical vagus nerve of anesthetized transgenic mice expressing ChR2 under cholinergic (ChAT) or glutamatergic (Vglut2) cell promoters. Lipopolysaccharide (3 mg/kg) was administered intraperitoneally to induce endotoxemia, and mice received optical VNS for 2 hours. Blood was collected 30 minutes after VNS termination and quantified via immunoassay for serum inflammatory cytokines (IL-6, IL-1{beta}, TNF-, IL-10) and C-reactive protein (CRP). ResultsChAT-selective optical VNS significantly reduced IL-6 (p = 0.027) and IL-1{beta} (p = 0.026) relative to Cre-negative sham controls. Vglut2-targeted stimulation did not significantly reduce IL-6, IL-1{beta}, or TNF- versus sham. Cytokine levels were significantly reduced with ChAT VNS compared to Vglut2 VNS in all pro-inflammatory cytokines (IL-6: p=0.024, IL-1{beta}: p=0.011, TNF-: p=0.030). The anti-inflammatory cytokine IL-10 was significantly decreased with ChAT versus Vglut2 VNS (p=0.033). CRP levels were not statistically different between groups. ConclusionsOptogenetic VNS targeting cholinergic neurons produced robust suppression of key pro-inflammatory cytokines IL-1{beta} and IL-6, whereas stimulation of glutamatergic neurons did not significantly alter inflammatory cytokine levels, highlighting the importance of pathway selectivity in the inflammatory effects of VNS. These findings highlight cell-type specific optogenetic neuromodulation as a valuable tool for assessing impact of vagal circuits and support preferential targeting of efferent cholinergic neurons in acute systemic inflammation.

Cell therapies hold transformative potential for treating cancer, neurologic disorders, organ failure, diabetes, and other conditions, but their widespread clinical deployment is constrained by the lack of scalable cryopreservation methods that maintain high post-thaw viability. Current standard practice using slow freezing can lead to cell death and impaired cell function. Vitrification offers an alternative by cooling samples rapidly enough to bypass ice formation entirely, better preserving cell structure and function. The high cooling and warming rates required for vitrification have previously been achieved by quenching microliter-scale samples directly into convective cooling and warming baths. Here, we present a cryopreservation platform that overcomes the throughput limitations of existing systems by combining a vibrating orifice aerosol generator with an impinging conical nozzle to generate and confine micrometer-scale droplets mid-flight in liquid nitrogen. This approach mitigates cooling losses due to the inverse Leidenfrost effect, increasing cooling and warming rates by nearly an order of magnitude compared to conventional droplet vitrification, while improving throughput by two orders of magnitude. To test the efficacy of this system, we cryoaerosolized and rewarmed human induced pluripotent stem cells, porcine red blood cells, and human dermal fibroblasts using only 190-25 wt% (2.5-3.7 M) permeating cryoprotective agent, achieving >90% post-thaw viability for HDFs and hiPSCs and 94% recovery for RBCs, with retained colony-forming capacity additionally demonstrated in hiPSCs. This work demonstrates the first scalable vitrification-based cryopreservation method capable of achieving both high cooling ({approx} 200, 000 K min-1) and warming rates ({approx} 1, 000, 000 K min-1) while maintaining the high-throughput processing required ([≥]100 mL h-1) for next-generation cell therapies. Significance StatementCell therapies require robust long-term storage methods to enable widespread clinical deployment. Current approaches utilizing refrigeration or small-scale vitrification cannot meet the scalability and viability requirements for next-generation therapeutics. In this work we demonstrate a cryoaerosolization process that achieves both ultra-rapid cooling rates (>200,000 {degrees}C min-1) and high throughput (>100 mL h-1) by generating micrometer-scale droplets and spraying in a liquid nitrogen impingement stream. Using only as little as 19 wt% cryoprotectant, we achieved >90% cell viability and maintained function, comparable to low-throughput methods, but at two orders of magnitude higher processing rates. We also introduce a just-in-time CPA loading approach that reduces toxicity exposure. This technique enables scalable vitrification-based cryopreservation of large-volume cell products.

Intrathecal (IT) drug delivery, i.e., the infusion of substances directly into cerebrospinal fluid (CSF) by lumbar, ventricular, or cisternal access points, is one method that can be used to bypass the blood brain barrier (BBB), however, IT-administered substances also suffer from rapid turnover and poor tissue penetration. Although nanoparticles and colloids can circulate within the subarachnoid space to sustain the levels of encapsulated drug in CSF, their access to deep tissue regions remains incomplete. Here, we present a new method for enhancing CNS delivery of IT-administered nanoparticles. CSF Flow Enhancement (CFE) refers to the manipulation of CSF production, distribution, and clearance for therapeutic purposes. We tested the overarching hypothesis that infusion of hypertonic fluid adjacent to the choroid plexus would enhance fluid production and movement to improve the CNS delivery of IT-administered nanoparticles. Model polystyrene nanoparticles (100nm) were solubilized in aCSF of increasing tonicity (1-9X tonicity) and infused into the cisterna magna, after which tissues were removed to examine delivery to CNS tissues and peripheral organs. Our results demonstrate that an infusion of up to 4X hypertonic aCSF in 10uL is well tolerated and yields significant improvements in CNS localization of co-administered nanoparticles, more than doubling the delivery of nanoparticles to the ventral surfaces of the brain and sometimes dramatic (up to 10-fold) increases in delivery to specific tissue regions and surfaces of the CNS. Significantly, we provide early evidence that modulation of tonicity can define the parenchymal fate of IT administered colloids: while nanoparticles were not detected in the brain parenchyma of mice that received a standard infusion, parenchymal delivery was observed for the 2X condition, and extensive perivascular infiltration of nanoparticles was observed for the 4X condition. Lastly, we show that the delivery improvements achieved by CFE are generalizable across multiple sizes of polystyrene nanoparticle (20, 40, or 100nm). Collectively, this work describes a tonicity-based approach for achieving CFE by the intrathecal route, which we posit is a useful and potentially generalizable approach for improving CNS drug delivery.

Background: Tumor necrosis factor- inhibitors such as infliximab and adalimumab have transformed autoimmune disease treatment; however, infliximab is a mouse-human chimeric antibody that remains immunogenic, is associated with self-association/aggregation liability, and requires prolonged intravenous administration. We humanized infliximab and engineered infliximab-derived candidates with improved potency and developability. Methods: Infliximab complementarity-determining regions were grafted onto human germline frameworks to generate humanized infliximab. STage-Enhanced Maturation (STEM) technology produced an affinity-matured clone (hInBG4), followed by targeted amino-acid substitutions in the complementarity-determining regions to generate LW2Y, LW2YR2S, and LW2YHR1K. Variants were evaluated by a cell-based tumor necrosis factor alpha neutralization assay, affinity-capture self-interaction nanoparticle spectroscopy, a baculovirus particle enzyme-linked immunosorbent assay, size-exclusion high-performance liquid chromatography, transient expression in human embryonic kidney 293 cells, and tumor necrosis factor alpha binding kinetics by biolayer interferometry, including dissociation at pH 7.4 and 5.8. Results: All three variants showed two- to three-fold higher neutralization potency than chimeric infliximab and outperformed adalimumab. Affinity-capture self-interaction nanoparticle spectroscopy shifts decreased from double-digit parental values to low single digits, while baculovirus particle binding ratios remained acceptable. Size-exclusion chromatography showed cleaner monomer peaks with reduced tailing, and expression increased relative to humanized infliximab. LW2Y combined very high affinity at pH 7.4 with markedly faster dissociation at pH 5.8, consistent with pH-dependent antigen release. Conclusions: Humanization, affinity maturation, and targeted complementarity-determining region re-engineering generated infliximab-derived candidates with improved potency and developability and identified LW2Y as a lead for further preclinical evaluation.

Synthetic grafts remain ineffective for small-caliber vascular applications due to thrombosis and intimal hyperplasia. To address these limitations, our lab designed a multilayer graft consisting of a hydrogel coating that promotes post-implantation endothelialization and an electrospun mesh that matches arterial mechanical properties. Damage-resistant hydrogels were engineered using a double-network system composed of polyether urethane diacrylamide and N-acryloyl glycinamide to enhance fracture resistance through hydrogen bonding. In this study, we utilized redox initiation to apply conformal, durable hydrogels to electrospun grafts. Bioactivity wa introduced using streptococcal collagen-like proteins containing 1{beta}1 and 2{beta}1 integrin-binding motifs, enabling selective cell-material interactions that support endothelialization while preserving acute thromboresistance. To establish the feasibility of these grafts as off-the-shelf devices, we evaluated coating integrity and bioactivity retention following sterilization and dynamic physiological loading. Sterilized composites exhibited surgically-associated damage resistance, indicating that sterilization did not compromise hydrogel durability. Coating integrity and bioactivity were also preserved after six weeks of physiological loading. Acut thromboresistance was supported by both static platelet adhesion assays and dynamic whole-blood bioreactor studies using heparinized blood, with low platelet adhesion observed relative to ePTFE. Finally, a pilot ovine carotid model demonstrated successful surgical handling and sustained graft patency. Collectively, these results highlight the promise of multilayer vascular grafts as durable, thromboresistant conduits for small-diameter vascular applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/741222v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@14ec910org.highwire.dtl.DTLVardef@17259e8org.highwire.dtl.DTLVardef@6cb611org.highwire.dtl.DTLVardef@1251d7d_HPS_FORMAT_FIGEXP M_FIG C_FIG

One barrier to developing an HIV-1 cure is viral reservoirs persisting within B cell follicles of lymphatic tissues, partly due to failure of HIV-specific cytotoxic cells to express the follicular-homing receptor CXCR5. Our group explores CAR cell therapies which also express CXCR5 as a potential cure strategy for HIV. Although previous studies have mostly explored CAR T cell therapies, CAR NK cells may be an attractive alternative as they can be used in allogeneic settings and are naturally cytotoxic towards HIV-infected cells. Here, we developed a novel and innovative multiplex engineering method for rhesus macaque NK cells to create virus-specific CAR NK cells multiplexed (MP) with CAR/CXCR5/IL-15/PD-1 KO/transient-CCR7. We first evaluated MP NK cells in vitro for functionality. MP NK cells were then infused into one chronically SIV-infected rhesus macaque to observe tolerance and localization of therapeutic cells. Finally, we performed a larger primate study in which SIV-infected rhesus macaques were infused with two doses of MP NK cells to study long-term localization, safety, and efficacy. In vitro, MP NK cells were expanded to clinically relevant numbers, migrated to chemokine signaling, and secreted cytotoxic cytokines in response to SIV-Env-expressing cells. In the preliminary rhesus macaque study, the therapy caused no adverse reactions, and CAR+ NK cells localized to sites of SIV replication within the spleen and lymph nodes. In the larger primate study, two doses of MP NK cells at 1.2 x 108 cells/kg were safe and increased the levels of NK cells and CAR+ NK cells found within lymphatic tissues. Importantly, the CAR+ NK cells detected in lymph nodes were predominantly CCR7+, demonstrating the importance of CCR7 and CXCR5 in combination for migration to SIV viral reservoirs in follicles of lymphatic tissues. This study is the first to demonstrate this type of complexity and combination of engineering techniques in NK cells. With further optimization, these techniques could lead to the development of novel NK cell therapies to treat HIV and other diseases.

Chronic, mechanically resilient thrombi remain difficult to remove rapidly and safely using existing therapies, which are limited by slow treatment speeds, reduced efficacy against aged clots and risks associated with embolic debris. Here we introduce Constrained Laser-Induced Cavitation (CLIC), a novel approach that confines laser-induced cavitation bubble generation and collapse within a miniaturized waveguide to enhance thrombolysis. Optimized CLIC removed retracted clots at a mass-loss rate of 393.5 mg/min, [~]40-fold higher than reported state-of-the-art sonothrombolysis under similar conditions. Systematic variation of channel length and laser parameters showed that CLIC efficacy depends strongly on treatment geometry and cavitation dynamics. Post-treatment analysis revealed cylindrical channels consistent with clot removal dominated by fluid jetting and suction-driven evacuation, with cavitation shockwaves likely contributing a secondary role. Debris fragment measurements remained predominantly below a 1 mm embolic-risk threshold, consistent with a promising embolic safety profile. These findings establish CLIC as a viable strategy for rapid thrombolysis of chronic, mechanically resistant thrombi.

Mesenchymal Stromal Cells (MSC) face several heterogeneity challenges hindering clinical and commercial success. Employing a multiple response model, interplay between donor heterogeneity, and critical processing parameters (CPPs), effects on MSC potency and cell expansion attributes were investigated through computed composite attribute scores. Twelve unique CPP combinations were tested in thirteen marrow-derived MSC(M) and five adipose-tissue MSC(AT) training and test datasets, respectively. Donor heterogeneity and select CPP conditions affected a curated gene panel (surrogate for MSC potency); while MSC expansion was primarily influenced by CPPs. Model performances were evaluated against clinical effectiveness data from a previously deployed clinical trial; top-performing model predicted donor rankings coincided with clinical effectiveness data, validating the modeling approach used. Our model predicted that only 8% of tested donors were agnostic to CPPs; a majority (62%) of donors showed CPP-dependent optimal composite quality attributes, with MSC seeding density as a key driver; medium supplementation and oxygen preferences were highly donor dependent. Approximately 30% of donors performed poorly at all conditions tested and may be prospectively identified using a subset of genes (TGFB, VEGF, PDCD1LG1, PDCD1LG2, IDO). Model predicted optimal parameters worked for 69% of tested donors, while sub-optimal parameters worked for only 23% of donors and were confirmed in an independent CD14+ macrophage assay. Our integrated computational and experimental framework predictably identified interactive effects of donor heterogeneity and CPP conditions to optimize MSC potency attributes.

Objective: To evaluate the effect of residual hair (RH) biomaterial particulates, biphasic electrical stimulation (ES), and their combination (RHES) on the kinetics and quality of skin wound healing. Method: Eighteen adult albino mice received bilateral, splinted 10-mm full-thickness dorsal excisional wounds and were randomly assigned to one of three animal groups producing four wound-level treatment conditions: untreated control (-) (n = 12), RH (n = 12), ES (n = 6), and combined RHES (n = 6 wounds). Daily wound images were segmented using an AI-assisted workflow: a U-Net (ResNet34 encoder, ImageNet-pretrained, trained on a parallel single-expert tracing study with held-out validation Dice = 0.906) generated initial boundary predictions, each reviewed and corrected as needed. Wound size measures (perimeter, area, equivalent diameter [D_eq], circularity, aspect ratio) were normalized to the day-0 value of each wound and analyzed by linear mixed-effects regression with mouse identity as a random intercept and mouse body weight as a covariate. On day 7, wounds were excised, fixed, processed for histology, and analyzed by Masson's trichrome (collagen content in granulation tissue) and GAP-43 immunohistochemistry (a marker of regenerative cellular activity). Results: All three treatments significantly accelerated wound closure compared to (-) (Day x Treatment interaction {chi}2(3) = 36.4, ***p < 0.0001). The closure-rate advantages on the log-D_eq scale were ES -0.047/day (***p < 0.0001), RHES -0.029/day (***p = 0.0005), and RH -0.022/day (**p = 0.0015). By day 7, mean D_eq had decreased to 0.58 of the day-0 value in ES, 0.69 in RHES, 0.73 in RH, and 0.79 in (-). Tissue analyses revealed treatment-specific differences in healing quality: RH and RHES wounds contained 6.1x and 8.5x more collagen in granulation tissue than (-) (both **p = 0.002 vs (-); both **p = 0.009 vs ES), and showed approximately 16x and 27x greater mean GAP-43 expression than (-), respectively; the RHES increase remained significant after Bonferroni correction (adjusted *p = 0.042), whereas the RH increase did not (adjusted p = 0.058). ES alone did not significantly increase either collagen content or GAP-43 expression. Wound shape was more circular and more stable across days in RH-containing groups. Mouse body weight did not predict closure, whereas image-derived dryness, eschar coverage, and wound contraction were significant negative predictors of measured wound size. Conclusion: ES, RH, and RHES each significantly improve wound closure kinetics. The improvement appears mechanistically distinct: ES principally accelerates closure rate, while RH principally enhances tissue-level regenerative markers (collagen deposition and GAP-43 expression). RHES combines both advantages.

Leptomeningeal disease (LD), involving the metastasis of cells to the leptomeningeal membranes in the central nervous system (CNS), can be a deadly complication of several different types of cancer originating in the periphery or CNS, including breast cancer (BC) and pediatric medulloblastoma (MB). Targeted therapy represents a promising new approach to improve overall survival for LD patients. To this date, angiopep-2 (Ang2) and transactivating transcriptional activator (TAT), two well-known peptides for their brain delivery capability, have been reported to transport therapeutic cargos into the CNS for treatment of disease. Current administration strategies, however, still rely on oral delivery or intravenous injection (IV), where the substances need to travel through complex biological barriers to reach the subarachnoid space (SAS), which is the primary location of LD. Our research group has focused on the intrathecal (IT) route of administration as an alternative approach that can potentially enable high exposure of drug to CSF exposed tissues. However, there is a major field gap in understanding how targeting peptides can access (or not access) LD as a function of their route of administration. Therefore, our work was focused on comparing the targeting capability of Ang2 vs TAT by IT vs IV routes of administration. We first generated two xenograft models of LD by directly infusing breast cancer cells (MDA-MB231) or medulloblastoma cells (HDMB03) into the SAS via intracisternal magna injection (ICM) to form BC-LD and MB-LD models, respectively. These tumor models were characterized for overall survival, tumor growth patterns, and presence of hydrocephalus. Second, we further administered fluorescently labeled Ang2 or TAT peptides either IV or ICM into tumor bearing mice. Neuraxial fluorescence images were examined to evaluate the targeting ability of these two peptides based on colocalization between peptide signal and tumor tissues ex vivo. We discovered that the median survival of both models was negatively related to the number of the cells infused. While HDMB03 cells tended to metastasize preferentially to the brain region, MDA-MB231 cells tended to metastasize preferentially to the spinal cord. Both models present hydrocephalus as one of the common clinical symptoms in LD patients. Compared to the healthy control, MB-LD yielded a 7.3-fold increase and BC-LD a 26.5-fold increase in ventricular volume. Furthermore, targeting achieved by TAT was significantly higher than targeting achieved by Ang2 in thoracic spine for the MB-LD model. For BC-LD model, TAT signal was found to be significantly higher than Ang2 signal in the olfactory bulbs, brain stem, thoracic spine, and lumbar spine regions. While both peptides showed a strong signal at 2 hours post ICM injection, signal was not detectable 24 hours after administration, reflecting washout or degradation. Significantly, these data provide evidence that ICM will be a preferable route of administration over IV for the purpose of maximally targeting LD.

Ex vivo airway engineering approaches such as 3D bioprinting offer a promising strategy for generating functional airway replacements, but the fabrication of hollow, patient-specific proximal airway constructs using translationally relevant bioinks remains challenging. This study describes the development of biocompatible, polymer-blended human airway-derived decellularized extracellular matrix (AW-dECM) bioinks for engineering structurally and mechanically relevant airway tissues. An optimal formulation consisting of 30 mg/mL AW-dECM and nanofibrillar cellulose alginate conjugated to RGD supported the bioprinting of simple and complex hollow airway structures with mechanical properties comparable to native airways ([~]8-10 kPa). The bioinks also promoted primary human airway epithelial cell viability, adhesion, and differentiation into mucociliary and secretory phenotypes during 28 days of air-liquid interface culture. Furthermore, subcutaneous implantation in immunocompetent rats demonstrated excellent biodegradative stability and overall biocompatibility over 30 days. Collectively, these findings establish a foundation for improved physiological airway models and future tissue-engineered airway replacements.

Inflammation-driven increases in nociception are prominent in pain pathologies associated with intervertebral disc (IVD) degeneration yet are difficult to model in vitro. Since neurons are exposed to multi-modal stimuli in vivo, it is critical for these exposures to be conserved in an in vitro test system. We developed a polydimethylsiloxane (PDMS)-based microfluidic platform to interrogate peripheral sensory neurons (SNs) in the presence of conditioned media from nucleus pulposus cells from the degenerated IVD, to model a potential impact of IVD cells secretome on pain sensing. Our platform enables controlled perfusion of cell-derived biochemical cues alongside a defined homogeneous electric field (EF) and supports real-time optical analysis. Computational modeling, fluid perfusion experiments, and conductivity measurements confirmed stable fluid transport and tunable homogeneous EF generation within the device. As proof of concept for neuronal stimulation, neuroblastoma (N2a) cells loaded with a fluorescent Ca2+ indicator exhibited a 56% increase in Ca2+ transient activity when exposed to media from degenerated IVDs, concomitant with increased IVD-derived IL-1{beta} production. Importantly, EF-stimulated Ca2+ transients increased in SNs derived from human induced pluripotent stem cells when exposed to conditioned media from primary human IVD cells, demonstrating the translation of this model system to human cells. Together, these results establish a versatile platform that enables controlled and simultaneous exposure to biochemical and electrical stimuli to quantify inflammation-driven peripheral neuronal hyperexcitability in tissue-neuron crosstalk.

Relapse remains a leading cause of treatment failure in acute myeloid leukaemia (AML), making haematopoietic stem cell transplantation (HSCT) the only curative option for many patients. Yet HSCT efficacy is often limited by impaired engraftment, driven by AML-induced remodelling of the bone marrow stem cell niche. Mesenchymal stromal cells (MSCs) are key mediators of niche formation and could, in principle, restore a supportive microenvironment when introduced alongside HSC therapy; but this strategy remains largely untested. A key obstacle to MSC-based therapy is that MSCs acquire a senescent, pro-inflammatory phenotype during standard in vitro expansion. We addressed this by engineering a polymer-laminin presentation system that suppresses senescence and preserves a proliferative, regenerative MSC phenotype during expansion. Then, to investigate potential cell therapy use, we developed a bioengineered in vitro model as a new approach methodology (NAM) for studying disease-driven niche modification. The system consists of MSC spheroids embedded in a synthetic hydrogel within a transwell platform, allowing controlled co-culture of healthy or AML-derived haematopoietic cells, therapeutic MSCs, and chemotherapeutic agents. Using this platform, we modelled an AML-like niche and showed that MSCs expanded via the polymer-laminin system, when introduced alongside HSCs, significantly improved HSCT engraftment relative to both standard-expanded MSCs and HSCT performed without MSC support. These results establish MSC phenotype maintenance as a critical determinant of therapeutic efficacy, and position this NAM as a platform for pre-clinical screening of niche-targeted therapies in AML.

Endothelial cells (ECs) derived from human induced pluripotent stem cells (hiPSCs) are increasingly used to model vascular diseases and test therapeutic strategies. However, the efficiency and reproducibility of differentiation can vary depending on the culture medium and its supplemented factors and stages. Here, we directly compared two defined media, APEL and BPEL, for iPSC-to-ECs differentiation. iPSCs were differentiated over 10 days with sequential growth factor induction, followed by magnetic-activated cell sorting or flow cytometry for CD31+ cells. Both media produced ECs with similar morphology and marker expression, including CD31 and VE-cadherin. Functional assays demonstrated comparable tube formation, indicating equivalent endothelial functionality. Cost analysis indicated that APEL had a higher total reagent cost but generated a higher total cell yield, resulting in a comparable cost per 10 total cells, whereas BPEL was more cost-efficient for producing CD31/VE-cadherin endothelial-specific cells. Our results suggest that APEL and BPEL media are equally effective for generating iPSC-derived ECs, providing flexibility in method selection for vascular disease modeling and drug discovery applications.

Treatment with chimeric antigen receptor (CAR) T cells has emerged as a promising immune therapy for relapsed and refractory hematologic malignancies. The CAR T cells are manufactured in a series of steps that involve isolating T cells from the patients leukapheresis product, genetically modifying them to express the CAR against the target antigen, and reinfusing them into the patient. Efficient T-cell enrichment from leukapheresis products is critical to the success of these therapies. Current methods for T-cell sorting on a clinical scale involve several washing steps to remove red blood cells and platelets, followed by T-cell selection and activation. These multi-step processes result in cell loss during processing and involve several handling steps. Here, we utilize fluidically assembled micromagnetic lenses to develop a high-throughput, continuous-flow microfluidic T-cell sorter, designated as the T-Chip, for sorting magnetic bead-labeled CD3+ T cells in a single step. Our approach allows direct sorting of T cells in expansion media from leukopaks without any washing steps, effectively removing 99.999% of RBCs and platelets from the leukapheresis product. A single 1-inch x 3-inch T-Chip can process leukapheresis product at a throughput of 60 mL/hr and 2.56 {+/-} 0.12 billion cells/hr. Using this optimized workflow, we demonstrate clinical-scale enrichment of highly pure CD3+ T cells (97.7 {+/-} 1.3%) with high viability (97.0 {+/-} 1.1%) and recovery (87.3 {+/-} 14.8%) in a functionally closed manner. Downstream processing of T cells isolated using the T-Chip yielded potent anti-mesothelin CAR T cells with demonstrated anti-tumor efficacy. Overall, by exploiting precisely engineered magnetic forces and laminar flow, the microfluidic T-Chip overcomes bottlenecks caused by low throughput and enables single-step large-scale T-cell purification for the rapid development of CAR T cells.

Gastrointestinal anastomotic leakage (AL) remains a life-threatening complication following gastrointestinal surgery, where outcomes critically depend on timely diagnosis. Current diagnostic strategies rely on periodic sampling and resource-intensive analysis in centralized laboratories. Here, we present a sterilizable, time-integrating hydrogel sensor platform for continuous, infrastructure-free monitoring of the patient's postoperative drain fluid. We introduce enzyme-responsive macromolecular networks for semi-quantitative bedside assessment of leak-associated digestive enzymes. The sensors retain functionality following lyophilization and ethylene oxide sterilization, enabling long-term storage and scalable deployment around the world. In a Swiss clinical cohort of 56 patients, including 19 with gastrointestinal anastomotic leakage, the sensor detected amylase-associated leaks two days (median) prior to clinical diagnosis with a sensitivity of 78% (95% CI 55-91) and a specificity of 95% (95% CI 82-99). The prospective validation in an independent cohort of 37 patients in Colombia, including seven patients with leaks, demonstrated 100% sensitivity (95% CI 64.6-100) and a 100% negative predictive value (95% CI 87.9-100.0), with sensor activation preceding standard clinical diagnosis by a median of five days. By converting episodic biochemical measurements into continuous, cumulative visual records, this infrastructure-free material platform enables close-meshed postoperative monitoring and may facilitate earlier recognition of anastomotic leakage across diverse healthcare settings.

Idiopathic pulmonary fibrosis is a devastating chronic lung disease characterized by progressive scarring of the lung, which leads to impaired gas exchange and ultimately death. While research has provided us with extensive understanding on end-stage disease, the factors that lead to forward-feedback loops of fibrotic progression are still not fully known. Cell intrinsic activation, pathological extracellular matrix (ECM) composition, and increased tissue stiffness are all hallmarks of advanced fibrosis, but the relative contribution of these factors to disease has been difficult to disentangle using classic in vivo models. In this study we created biomaterials-based 3D lung models that incorporate geometrically relevant co-culture of lung epithelial cells and fibroblasts with tunable stiffness, ECM-containing hybrid-hydrogels. Using this model system, we demonstrated that environmental stiffness has the strongest effect on overall fibroblast activation. RNAseq analysis revealed unique gene-level changes in both fibroblasts and epithelial cells due to both composition and stiffness, highlighting the importance of incorporating both factors into any in vivo disease models. Overall, these results reinforce the value of biomaterials-based models in understanding disease pathogenesis, and their potential for screening of treatment responses.