Intracellular pathogens, such as Listeria monocytogenes (LM), manipulate host cells to spread from the initial infection site to distant organs through the bloodstream. For that, LM hijacks mononuclear phagocytes to traverse vascular endothelial cell (EC) linings, but how transmigration is regulated by ECs is poorly understood. Here, we show that LM infection profoundly alters EC biomechanical responses to macrophages (MΦs). Videomicroscopy revealed that EC-MΦ contact induces EC polarization, alignment, and reduced motility. However, only interactions with uninfected MΦs increased EC traction and monolayer stresses and barrier integrity. This biomechanical response is largely contact-dependent and significantly attenuated during infection, thus contributing to the enhanced rate of LM-infected MΦ transmigration. Consistently, in the zebrafish model, infection increased endothelial permeability and phagocyte extravasation. These findings reveal that LM infection overrides MΦ-induced endothelial barrier strengthening to promote pathogen dissemination, a biomechanical strategy that could be harnessed for infection control.

The pioneering discovery by Yamanaka and colleagues enabling the reprogramming of terminally differentiated somatic cells into induced pluripotent stem cells (iPSCs) has opened transformative opportunities for disease modeling and regenerative medicine, particularly in the context of inherited monogenic disorders. Patient-specific iPSCs can be generated, expanded almost indefinitely, and differentiated into a broad spectrum of cell types, including hematopoietic stem and progenitor cells, mature myeloid cells, and leukemic cells. Despite important limitations - such as epigenetic memory, variable differentiation efficiency, and concerns regarding tumorigenicity - iPSCs have become an indispensable experimental platform for studying inherited hematological disorders and malignancies, providing a renewable and physiologically relevant source of cells for downstream analyses. Beyond their research applications, iPSC-derived blood cells are increasingly being explored in preclinical studies and early-phase clinical trials as potential therapeutic products. The advent of CRISPR/Cas9 genome editing, pioneered by Charpentier and Doudna, has further advanced iPSC-based models by enabling precise correction or introduction of disease-causing mutations and the generation of isogenic control lines. This approach facilitates detailed mechanistic studies of defective hematopoiesis, enables drug discovery and repurposing through in silico screening platforms - such as L1000CDS2 and the Connectivity Map - and supports preclinical therapeutic validation. In this review, we summarize key applications of iPSC technology in hemato-oncology, discuss its major advantages and current limitations, and highlight emerging directions, including scalable iPSC-derived blood cell therapies for inherited and acquired bone marrow failure syndromes and leukemia.

Abstract The peritoneum, the body's largest serous membrane, plays critical roles in abdominal homeostasis and immune defense. When disrupted by surgery or disease, it can lead to devastating complications including peritoneal adhesions—affecting up to 93% of surgical patients—peritonitis, and metastatic spread. Current research models fail to capture the complexity of human peritoneal biology, relying on inadequate animal models or oversimplified 2D cultures. Here, we introduce a PDMS‐free microfluidic platform that recreates the structural and functional architecture of human peritoneum. Our system combines immortalized mesothelial cells (MeT5A) with patient‐derived peritoneal fibroblasts in a physiologically relevant 3D environment, enabling real‐time analysis of peritoneal function and dysfunction. Through systematic evaluation of stromal matrices, we identified fibrin gel as optimal for supporting healthy mesothelial monolayer formation while maintaining excellent cell viability over 14 days. Importantly, we demonstrate the platform's translational potential by successfully modeling peritoneal adhesion formation. This innovative tool may improve the understanding of peritoneal biology, accelerating drug discovery and developing personalized treatment strategies for peritoneal diseases.

Intravitreal (IVT) administration of adeno-associated virus (AAV) vectors is considered a promising strategy for retinal gene therapy. Random peptide-display-based directed evolution has yielded novel AAV capsids with potent retinal bioactivity. However, recent reports on safety risks following IVT AAV administration indicate that significant opportunities remain to further refine AAV vector technology. Here, we performed iterative in vivo NGS-guided screening of AAV2 peptide-display libraries in both mice and non-human primates (NHPs), integrating barcoded functional analytics, and single-nuclei RNA sequencing to identify retina-tropic AAV variants. The top-performing capsid in mice, AAV2-GAYPKSP robustly drove eGFP expression in a human retina-on-chip model but failed to drive gene expression in the NHP retina following IVT delivery. In NHPs, a barcoded evaluation of 70 retina-tropic variants revealed a complex correlation between viral genome enrichment and transgene transcriptional output, providing insights that enhance the mechanistic interpretation and methodological design of AAV library selection strategies. Nonetheless, several NHP variants, including AAV2-I.1 and AAV2-I.12, demonstrated broad retinal transduction and potent expression, outperforming benchmarks AAV2 and AAV2-7m8. Single-nucleus RNA sequencing confirmed broad cell-type tropism of the top performing variants. Collectively, these findings expand the AAV toolkit for retinal gene therapy and underscore the importance of optimized screening methodologies in vector discovery.

Reliable modeling of human adaptive immune responses is a prerequisite to understand processes leading to vaccine-induced protective immunization, to overcome the poor predictive value of non-clinical in vivo and in vitro models and to drive informed decisions in vaccine development pipelines. Here, we present a centrifugal microfluidics-based organ-on-chip approach to generate an organotypic high density lymphoid-tissue-on-chip. The model enables long-term culture of lymphoid tissue while preventing autoactivation and shows raised antigen-specific antibody responses against influenza vaccines for up to 4 weeks on-chip. Antibody response of different magnitude and quality could be induced both by direct antigen exposure as well as by recruitment of peripheral antigen-presenting cells. The model represents an attractive approach to evaluate the impact of the mode of antigen delivery on adaptive immune responses. Beyond applications in vaccine development, the lymphoid-tissue-on-chip provides a platform to study cellular interactions during homeostasis, immune responses, and drug treatment over several weeks.

Accurate prediction of the hepatic clearance in humans is essential during pre-clinical drug development. As conventional in vitro models do not replicate the complexity of the human liver physiology, hepatocyte-specific functions are rapidly lost resulting in limited assay sensitivity. In this study, a commercially available organ-on-chip model featuring a two-compartment microfluidic architecture was evaluated to estimate human hepatic clearance for a panel of 15 commercial drug compounds with diverse metabolic pathways and clearance rates. Upon further optimization of the model for the co-culture of primary human hepatocytes and liver sinusoidal endothelial cells, key system parameters - including compound permeability, non-specific binding, and evaporation - were systematically characterized. Despite optimized conditions, the human hepatic clearance was generally underpredicted by the liver-chip. The best performance was observed for compounds with low to moderate clearance while the underprediction was more pronounced for compounds exhibiting high metabolic turnover. By introducing a model-specific systematic scaling factor, more than 79% of clearance predictions fell within a three-fold range of observed human values. The study identified challenges in liver-chip systems for ADME applications that stem from specific design features and proposes optimization strategies for using liver-chips in metabolic stability assessments.

Human skeletal muscle is the principal site of insulin-stimulated glucose disposal and a major mediator of exercise-induced metabolic benefits, yet human models that preserve metabolic and exercise responsiveness remain limited. We generated primary human skeletal muscle organoids from donor-derived CD56+ myoblasts using a collagen-based extracellular matrix and serum-free IGF1-guided differentiation. The organoids formed aligned contractile tissues containing oxidative and glycolytic fiber type-like myotubes, displayed enhanced mitochondrial respiration, insulin-stimulated glucose uptake, and reproducible force generation. Electrical pulse stimulation induced AMPK activation, increased glucose utilization and lactate production, and upregulated canonical exercise-responsive genes including NR4A3 and PPARGC1A. Notably, transcriptional responses to in vitro exercise overlapped with acute exercise responses observed in skeletal muscle biopsies from the same donors. The organoids further detected functional impairments of skeletal muscle performance induced by TGF-β1 and metformin and increased speed generation by testosterone treatment. These findings establish a donor-specific human skeletal muscle platform that recapitulates key features of insulin action and exercise adaptation and may enable mechanistic studies of skeletal muscle metabolism, exercise responsiveness, and therapeutic interventions relevant to diabetes.

Accurate predictions of complex clinical drug-drug interactions (DDIs), arising from dual induction and time-dependent inhibition (TDI) of CYP3A4, has remained challenging with conventional in vitro and static/dynamic modeling approaches. In this work, we aimed to anticipate the hepatic DDI effects of 6 CYP3A4 precipitant drugs using a liver-chip coupled to microfluidic perfusion, which enabled simulating clinically relevant pharmacokinetic (PK) time-concentration profiles. Midazolam clearance was measured in the liver-chip to determine the CYP3A4-mediated DDI net effect following exposure to precipitants under either dynamic or constant concentration conditions. For direct comparison, hepatic DDI reference values were generated based on clinical DDI studies by physiologically based PK modeling. Under microfluidic perfusion, CYP3A4 activity in the liver-chip was retained for 3 days and inducible by rifampicin. Although most precipitant drugs induced CYP3A4 mRNA levels, CYP3A4 activity net effects showed either induction or inhibition, in line with clinical observations. Compared with a mechanistic static net effect model and physiologically based PK simulations, liver-chip predictions showed closer alignment and higher accuracy relative to hepatic reference values. Although constant exposures offered the strongest quantitative performance, the ability to apply dynamic PK profiles represents a distinctive feature of this platform. Collectively, these findings position the human liver-chip as a novel translational platform for predicting complex hepatic CYP3A4 DDIs arising from dual TDI and induction in a single, holistic in vitro model. This approach aligns with the broader global regulatory shift toward human-relevant new approach methodologies. SIGNIFICANCE STATEMENT: Complex CYP3A4-mediated drug-drug interactions (DDIs), driven by concurrent time-dependent inhibition and induction, remain difficult to anticipate using conventional in vitro and modeling frameworks. This study establishes a human liver-chip model capable of capturing dual CYP3A4 inhibition and induction within a single experiment, and evaluates how clinical exposure regimes shape the quantitative prediction of hepatic DDI effects. Relative to modeling approaches, this platform demonstrates superior accuracy against hepatic DDI references, supporting more translational DDI risk assessments.

Abstract Background Ovarian cancer (OvCa) ranks as the most lethal gynecological malignancy in women worldwide. This complex disease, which can develop independently of a woman’s age, is characterized by late diagnosis, pronounced tumor heterogeneity, and an immunosuppressive tumor microenvironment (TME). Incremental diagnostic tools that could better inform clinicians on potential therapy resistance or subsets of patients that could benefit from new drug modalities represent a critical unmet need to improve patient care and potentially the identification of new biomarkers. Objective This study aimed to establish a reconfigurable patient-derived OvCa-on-chip platform for longitudinal functional profiling of tumor cell death, immune activation, and patient-specific responses to TIL-mediated killing, PD-L1 blockade, and sequential chemo-immunotherapy. Methods Patient-derived OvCa microtumors (PDM) were integrated with sequential integration of autologous tumor-infiltrating lymphocytes (TILs) into a perfusable microfluidic chip in the presence of different single and combination treatment regimens of chemotherapy and immune checkpoint inhibitors (ICIs). Treatment responses were assessed by longitudinal quantification of caspase-cleaved cytokeratin 18 (ccCK18) as marker of apoptotic epithelial tumor cell death, as well as cytokine/chemokine release in chip effluents, and multiplex flow cytometry-based characterization of autologous TIL subsets. Results The perfusable OvCa-on-chip platform supported long-term culture of PDM while maintaining key structural and microenvironmental features of the primary tumor. Multidimensional analyses, including time-resolved assessment of tumor cell death, secretome profiling and correlative analysis of autologous TIL subsets revealed patient-specific tumor-immune response patterns and heterogenous sensitivity to TIL-mediated killing, PD-L1 blockade, and sequential chemo-immunotherapy. Correlation analyses identified treatment-dependent associations between specific TIL phenotypes and functional tumor cell killing. PD-1-expressing CD4⁺ TIL subsets correlated with enhanced tumor cell killing, whereas terminally exhausted CD8⁺PD-1⁺Tcf1⁻ TILs negatively correlated with durvalumab responses. In contrast, tumor-reactive CD8⁺CD39⁺ TILs were associated with improved responses under sequential chemo-immunotherapy conditions. Conclusion Collectively, this OvCa-on-chip system represents a complex in vitro model (CIVM) that combines 3D tumor tissue with autologous immune cells in a microfluidic platform. Resembling a physiologically relevant human preclinical platform, it allows for the time-resolved functional assessment of patient-specific responsiveness to OvCa therapies, with direct implications for personalized treatment stratification.

Tumor tissue engineering, integrating organoid, microfluidic, and biofabrication technologies, has opened new avenues for cancer research. Leveraging advanced bioengineering and biomaterials, these 3D models capture tumor architecture, cellular heterogeneity, biomechanics, and biochemical characteristics for disease modeling. Despite recognition that tissue organization influences malignancy and drug resistance, clinically oriented 3D approaches are rare, largely due to tumor microenvironment complexity, cellular plasticity, and interpatient heterogeneity. With a primary emphasis on gastrointestinal malignancies, we outline the capabilities and remaining limitations of organoid-based cancer models, including developmentally defined stem cell-derived systems that enable controlled early-stage modeling when premalignant material is scarce. We discuss patient-derived organoids as clinical avatars for therapy response prediction and summarize recent clinical trials that delineate key bottlenecks hindering routine implementation. Finally, we outline how innovations in biomaterial design, biofabrication, and microfluidics, benchmarking against patient data, and artificial intelligence are converging to better reconstruct tumor complexity, improve experimental tractability, and accelerate translation.

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.

Adipose tissue is increasingly recognised as a central regulator of systemic metabolism. Beyond energy storage, adipose tissue integrates nutrient sensing, endocrine signalling, and immune responses, and actively communicates with other organs to coordinate metabolic homeostasis. This functional complexity arises from the coordinated activity of adipocytes and stromal cells, whose interactions dynamically regulate both physiological and pathological states. Most in vitro experimental models used in drug development and mechanistic research have simplified this complexity, whereas in vivo models integrate systemic physiology but lack human specificity. This mismatch limits the ability to predict adipose-specific drug effects, particularly for compounds targeting metabolic pathways, inflammation, or inter-organ signalling. In pharmacological contexts this limitation is particularly relevant because adipose tissue not only acts as a therapeutic target but also influences drug distribution, bioavailability, and efficacy through lipid partitioning, endocrine signalling, and immune modulation. Models that fail to capture these features risk overlooking key mechanisms of action or mispredicting therapeutic outcomes. Microphysiological systems that reconstruct adipose tissue complexity offer a framework to bridge this gap. By integrating multiple adipose-relevant cell types, such as mature adipocytes and stromal vascular fraction cells, along with physiological perfusion and controllable microenvironments, these systems could help address how cell-cell interactions shape metabolic function and pharmacological responses. As such, they represent a translational platform to interrogate drug mechanisms, evaluate tissue-specific efficacy, and could become tools to predict systemic pharmacological effects in metabolically relevant human settings.

Intranasal administration represents a safe and non-invasive route for drug delivery to the brain; however, clinical translation remains limited due to anatomical and physiological barriers. We present a modular hybrid biomaterial platform (NanoInBrain) that bypasses the blood-brain barrier via the olfactory route and enables central nervous system (CNS) drug delivery. The platform integrates a rationally designed polypeptide-based nanocarrier with a depot-forming hydrogel vehicle - a hyaluronic acid-poly-L-glutamate crosspolymer (HA-CP, Yalic®) - adapted from dermatological applications to enhance nasal mucosal retention and brain uptake. We engineered the nanocarrier system using star-shaped poly-L-glutamate (StPGA) architectures and systematically tuned physicochemical properties to optimize mucosal interaction and CNS diffusion. We introduced mucoadhesive and mucodiffusive functionalities via C-terminal odorranalectin (OL) conjugation, which improved nasal epithelium permeation through receptor-mediated mechanisms. Redox-responsive disulfide crosslinking (StPGA-CL-SS) further enhanced mucosal transport by enabling thiol-mediated anchoring to mucin glycoproteins, outperforming inert click-crosslinked variants. Ex vivo Franz diffusion studies and a nasal-mucosa-on-chip model demonstrated robust permeation, with in vivo imaging confirming brain distribution and intracellular uptake in neurons and microglia. Incorporation of HA-CP prolonged nasal residence (∼4 h) and increased total brain accumulation while being well-tolerated. This new platform combines architectural tunability, bioresponsive surface chemistry, and depot-mediated delivery in a scalable, biocompatible nose-to-brain delivery system with potential for treating neurological disorders.

Pancreatic ductal adenocarcinoma (PDAC) remains a major clinical challenge due to late detection and limited treatment responsiveness. To better evaluate complex immunotherapies in a human-relevant setting, we developed an integrated organoid-immune co-culture pipeline using PDAC patient-derived organoids (PDOs) and matched HLA immune cells. As a proof of concept, we assessed an MSLN-targeted nanovaccine (Mesovac), alone and in combination with FOLFIRINOX chemotherapy and Atezolizumab. We evaluated Mesovac across a multi-stage pipeline, including T-cell stimulation, ex vivo expansion, and PDO-immune co-cultures, to assess immune activation, specificity, and synergy with combinatorial treatments. MSLN-stimulated T-cells, derived from PDAC patients, showed increased IFN-γ production and selective infiltration into MSLN-expressing PDOs. Artificial antigen-presenting cells (aAPCs) boosted the expansion of reactive T-cells, enhancing antitumor responses. Notably, combining Mesovac with FOLFIRINOX and Atezolizumab maintained PD-L1+ T-cell levels and reduced cancer stem cells and aggressive PDAC subsets. Using this advanced in vitro workflow, we highlight that this platform, using human organoid-immune cell co-cultures, enables the evaluation of complex processes related to nanovaccine strategies that would not be possible in vivo.

Multi-organ-chip (MOC) models provide a plethora of auspicious opportunities to replace current in vitro and in vivo models for a more systemic investigation of human (patho-)physiology for drug development and personalized medicine. Integration of individual organ tissues into a systemic circulation remains a major challenge for their implementation/application. Modular ‘mix-and-match’ connection strategies are beneficial in their flexibility for individual organ-on-chip (OoC) module designs, and their connection and experimental timelines, but yet lack a facile implementation/realization without the addition of external connectors and dead volume. We introduce a novel concept for the flexible plug and play integration of OoC modules to an MOC platform by integrated µGaskets. The thermoplastic elastomer (TPE)-based µGaskets provide a highly robust and simultaneously easy connection of customizable tissue models. We characterized the facile fabrication of connection chips equipped with µGaskets and proved their functionality and durability in different burst, pressure and reusability tests.

In the past decades, vaccine development has made great strides. Nevertheless, more often than not, vaccine candidates fail in advanced stages of development and clinical trials. A key reason is the poor predictive value of non-clinical in vivo and in vitro models, due to either species-specific differences in the immune response or insufficient reflection of physiological vaccine mechanisms. Reliable modeling of human adaptive immune responses is a prerequisite to understand processes leading to vaccine-induced protective immunization and to drive informed decisions in vaccine development pipelines. Here, we present a centrifugal microfluidics based organ-on-chip approach to generate an organotypic high density lymphoid tissue on-chip. The model enables long-term culture of lymphoid tissue and raised antigen-specific antibody responses against influenza vaccines even after four weeks on-chip. Antibody response of different magnitude and quality could be induced both by direct antigen exposure as well as by recruitment of antigen-presenting cells from the periphery. The model represents an attractive approach to evaluate the impact of the mode of antigen delivery on adaptive immune responses. Beyond applications in vaccine development, the lymphoid-tissue-on-chip provides a platform to study cellular interactions during homeostasis, immune responses and long-term impact of immunomodulators over several weeks. ### Competing Interest Statement The authors have declared no competing interest.

Mutations in the tripeptidyl peptidase 1 (TPP1) gene lead to neuronal ceroid lipofuscinosis type 2 (CLN2), characterized by lysosomal accumulation of lipofuscins predominantly in the brain and retina. The ocular phenotype is characterized by outer retinal degeneration that leads to vision loss. Leveraging human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs), retinal pigmented epithelial cells, and the retina-on-chip system, we establish an in vitro CLN2 model that recreates the principal histological hallmarks, namely the accumulation of subunit C of mitochondrial ATP synthase (SCMAS) and lipids mainly in the outer retina. Furthermore, single-cell RNA sequencing reveals a dysregulation of translational and mitochondrial function in CLN2 cones. Finally, adeno-associated virus (AAV)-mediated TPP1 gene therapy can restore TPP1 expression and decrease and even prevent SCMAS accumulations. Our study uses an innovative human-relevant microphysiological retinal disease models, uncovers previously uncharacterized mechanisms of CLN2 pathophysiology, and demonstrates the potential of AAV9.hCLN2 gene therapy for CLN2 disease, potentially treating patient blindness.