IGF1 promotes human myotube differentiation toward a mature metabolic and contractile phenotype.

Skeletal muscle mediates the beneficial effects of exercise, thereby improving insulin sensitivity and reducing the risk for type 2 diabetes. Current human skeletal muscle models in vitro are incapable of fully recapitulating its physiological functions especially muscle contractility. By supplementation of insulin-like growth factor 1 (IGF1), a growth factor secreted by myofibers in vivo, we aimed to overcome these limitations. We monitored the differentiation process starting from primary human CD56-positive myoblasts in the presence/absence of IGF1 in serum-free medium in daily collected samples for 10 days. IGF1-supported differentiation formed thicker multinucleated myotubes showing physiological contraction upon electrical pulse stimulation (EPS) following day 6. Myotubes without IGF1 were almost incapable of contraction. IGF1 treatment shifted the proteome toward skeletal muscle-specific proteins that contribute to myofibril and sarcomere assembly, striated muscle contraction, and ATP production. Elevated PPARGC1A, MYH7, and reduced MYH1/2 suggest a more oxidative phenotype further demonstrated by higher abundance of proteins of the respiratory chain and elevated mitochondrial respiration. IGF1-treatment also upregulated glucose transporter (GLUT)4 and increased insulin-dependent glucose uptake compared with myotubes differentiated without IGF1. To conclude, addition of IGF1 to serum-free medium significantly improves the differentiation of human myotubes that showed enhanced myofibril formation, response to electrical pulse stimulation, oxidative respiratory capacity, and glucose metabolism overcoming limitations of previous standards. This novel protocol enables investigation of muscular exercise on a molecular level.NEW & NOTEWORTHY Human skeletal muscle models are highly valuable to study how exercise prevents type 2 diabetes without invasive biopsies. Current models did not fully recapitulate the function of skeletal muscle especially during exercise. By supplementing insulin-like growth factor 1 (IGF1), the authors developed a functional human skeletal muscle model characterized by inducible contractility and increased oxidative and insulin-sensitive metabolism. The novel protocol overcomes the limitations of previous standards and enables investigation of exercise on a molecular level.

Breast cancer-on-chip for patient-specific efficacy and safety testing of CAR-T cells.

Physiologically relevant human models that recapitulate the challenges of solid tumors and the tumor microenvironment (TME) are highly desired in the chimeric antigen receptor (CAR)-T cell field. We developed a breast cancer-on-chip model with an integrated endothelial barrier that enables the transmigration of perfused immune cells, their infiltration into the tumor, and concomitant monitoring of cytokine release during perfused culture over a period of up to 8 days. Here, we exemplified its use for investigating CAR-T cell efficacy and the ability to control the immune reaction with a pharmacological on/off switch. Additionally, we integrated primary breast cancer organoids to study patient-specific CAR-T cell efficacy. The modular architecture of our tumor-on-chip paves the way for studying the role of other cell types in the TME and thus provides the potential for broad application in bench-to-bedside translation as well as acceleration of the preclinical development of CAR-T cell products.

Recommendations on fit-for-purpose criteria to establish quality management for microphysiological systems and for monitoring their reproducibility.

Cell culture technology has evolved, moving from single-cell and monolayer methods to 3D models like reaggregates, spheroids, and organoids, improved with bioengineering like microfabrication and bioprinting. These advancements, termed microphysiological systems (MPSs), closely replicate tissue environments and human physiology, enhancing research and biomedical uses. However, MPS complexity introduces standardization challenges, impacting reproducibility and trust. We offer guidelines for quality management and control criteria specific to MPSs, facilitating reliable outcomes without stifling innovation. Our fit-for-purpose recommendations provide actionable advice for achieving consistent MPS performance.

Microphysiological pancreas-on-chip platform with integrated sensors to model endocrine function and metabolism.

Pancreatic in vitro research is of major importance to advance mechanistic understanding and development of treatment options for diseases such as diabetes mellitus. We present a thermoplastic-based microphysiological system aiming to model the complex microphysiological structure and function of the endocrine pancreas with concurrent real-time read-out capabilities. The specifically tailored platform enables self-guided trapping of single islets at defined locations: ß-cells are assembled to pseudo-islets and injected into the tissue chamber using hydrostatic pressure-driven flow. The pseudo-islets can further be embedded in an ECM-like hydrogel mimicking the native microenvironment of pancreatic islets in vivo. Non-invasive real-time monitoring of the oxygen levels on-chip is realized by the integration of luminescence-based optical sensors to the platform. To monitor insulin secretion kinetics in response to glucose stimulation in a time-resolved manner, an automated cycling of different glucose conditions is implemented. The model's response to glucose stimulation can be monitored via offline analysis of insulin secretion and via specific changes in oxygen consumption due to higher metabolic activity of pseudo-islets at high glucose levels. To demonstrate applicability for drug testing, the effects of antidiabetic medications are assessed and changes in dynamic insulin secretion are observed in line with the respective mechanism of action. Finally, by integrating human pancreatic islet microtissues, we highlight the flexibility of the platform and demonstrate the preservation of long-term functionality of human endocrine pancreatic tissue.

Cancer-mediated axonal guidance of sensory neurons in a microelectrode-based innervation MPS.

Despite recent advances in the field of microphysiological systems (MPSs), availability of models capable of mimicking the interactions between the nervous system and innervated tissues is still limited. This represents a significant challenge in identifying the underlying processes of various pathological conditions, including neuropathic, cardiovascular and metabolic disorders. In this novel study, we introduce a compartmentalized three-dimensional (3D) coculture system that enables physiologically relevant tissue innervation while recording neuronal excitability. By integrating custom microelectrode arrays into tailored glass chips microfabricated via selective laser-etching, we developed an entirely novel class of innervation MPSs (INV-MPS). This INV-MPS allows for manipulation, visualization, and electrophysiological analysis of individual axons innervating complex 3D tissues. Here, we focused on sensory innervation of 3D tumor tissue as a model case study since cancer-induced pain represents a major unmet medical need. The system was compared with existing nociception models and successfully replicated axonal chemoattraction mediated by nerve growth factor (NGF). Remarkably, in the absence of NGF, 3D cancer spheroids cocultured in the adjacent compartment induced sensory neurons to consistently cross the separating barrier and establish fine innervation. Moreover, we observed that crossing sensory fibers could be chemically excited by distal application of known pain-inducing agonists only when cocultured with cancer cells. To our knowledge, this is the first system showcasing morphological and electrophysiological analysis of 3D-innervated tumor tissuein vitro, paving the way for a plethora of studies into innervation-related diseases and improving our understanding of underlying pathophysiology.

Immunocompetent PDMS-Free Organ-on-Chip Model of Cervical Cancer Integrating Patient-Specific Cervical Fibroblasts and Neutrophils.

Despite preventive measures and available treatments, cervical cancer still ranks as the fourth most prevalent cancer among women worldwide and remains the leading cause of cancer death in women in many developing countries. To gain further insights into pathogenesis and to develop novel (immuno)therapies, more sophisticated human models recreating patient heterogeneities and including aspects of the tumor microenvironment are urgently required. A novel polydimethylsiloxane-free microfluidic platform, designed specifically for the generation and ccultivation of cervical cancerous tissue, is introduced. The microscale open-top tissue chambers of the cervical cancer-on-chip (CCoC) enable facile generation and long-term cultivation of SiHa spheroids in co-culture with donor-derived cervical fibroblasts. The resulting 3D tissue emulates physiological architecture and allows dissection of distinct effects of the stromal tissue on cancer viability and growth. Treatment with cisplatin at clinically-relevant routes of administration and dosing highlights the platform's applicability for drug testing. Moreover, the model is amenable for integration and recruitment of donor-derived neutrophils from the microvasculature-like channel into the tissue, all while retaining their ability to produce neutrophil extracellular traps. In the future, the immunocompetent CCoC featuring donor-specific primary cells and tumor spheroids has the potential to contribute to the development of new (immuno)therapeutic options.

Developer's Guide to an Organ-on-Chip Model.

Over the past decade, organ-on-chip research has been one of the most prolific areas of the entire field of tissue engineering. The development of organ-on-chip models requires an integrated interdisciplinary approach merging technologies and concepts from several different disciplines, including microfabrication, microfluidics, biomaterials, stem cell science, pharma-/toxicology, and medicine. In this perspective, we follow the journey of an organ-on-chip through its many different stages, from (i) the initial idea/specific scientific question to (ii) the design/concept phase, (iii) the engineering (fabrication and materials, sensor/actuator integration) and (iv) biology considerations (cell sources, biomaterials/scaffold), (v) the cell injection and tissue assembly process, (vi) the assay development, and (vii) the functional validation, all the way to (viii) the final applications. By summarizing some of the key learnings and findings from a developer's perspective and identifying suitable introductory reviews, this perspective strives to provide a conceptual, stepwise guide for the holistic development of an organ-on-chip model.

Fusing spheroids to aligned µ-tissues in a heart-on-chip featuring oxygen sensing and electrical pacing capabilities.

Over the last decade, Organ-on-Chip (OoC) emerged as a promising technology for advanced in vitro models, recapitulating key physiological cues. OoC approaches tailored for cardiac tissue engineering resulted in a variety of platforms, some of which integrate stimulation or probing capabilities. Due to manual handling processes, however, a large-scale standardized and robust tissue generation, applicable in an industrial setting, is still out of reach. Here, we present a novel cell injection and tissue generation concept relying on spheroids, which can be produced in large quantities and uniform size from induced pluripotent stem cell-derived human cardiomyocytes. Hydrostatic flow transports and accumulates spheroids in dogbone-shaped tissue chambers, which subsequently fuse and form aligned, contracting cardiac muscle fibers. Furthermore, we demonstrate electrical stimulation capabilities by utilizing fluidic media connectors as electrodes and provide the blueprint of a low-cost, open-source, scriptable pulse generator. We report on a novel integration strategy of optical O2 sensor spots into resin-based microfluidic systems, enabling in situ determination of O2 partial pressures. Finally, a proof-of-concept demonstrating electrical stimulation combined with in situ monitoring of metabolic activity in cardiac tissues is provided. The developed system thus opens the door for advanced OoCs integrating biophysical stimulation as well as probing capabilities and serves as a blueprint for the facile and robust generation of high density microtissues in microfluidic modules amenable to scaling-up and automation.

Microphysiological stem cell models of the human heart.

Models of heart disease and drug responses are increasingly based on human pluripotent stem cells (hPSCs) since their ability to capture human heart (dys-)function is often better than animal models. Simple monolayer cultures of hPSC-derived cardiomyocytes, however, have shortcomings. Some of these can be overcome using more complex, multi cell-type models in 3D. Here we review modalities that address this, describe efforts to tailor readouts and sensors for monitoring tissue- and cell physiology (exogenously and in situ) and discuss perspectives for implementation in industry and academia.

Autologous Human Immunocompetent White Adipose Tissue-on-Chip.

Obesity and associated diseases, such as diabetes, have reached epidemic proportions globally. In this era of "diabesity", white adipose tissue (WAT) has become a target of high interest for therapeutic strategies. To gain insights into mechanisms of adipose (patho-)physiology, researchers traditionally relied on animal models. Leveraging Organ-on-Chip technology, a microphysiological in vitro model of human WAT is introduced: a tailored microfluidic platform featuring vasculature-like perfusion that integrates 3D tissues comprising all major WAT-associated cellular components (mature adipocytes, organotypic endothelial barriers, stromovascular cells including adipose tissue macrophages) in an autologous manner and recapitulates pivotal WAT functions, such as energy storage and mobilization as well as endocrine and immunomodulatory activities. A precisely controllable bottom-up approach enables the generation of a multitude of replicates per donor circumventing inter-donor variability issues and paving the way for personalized medicine. Moreover, it allows to adjust the model's degree of complexity via a flexible mix-and-match approach. This WAT-on-Chip system constitutes the first human-based, autologous, and immunocompetent in vitro adipose tissue model that recapitulates almost full tissue heterogeneity and can become a powerful tool for human-relevant research in the field of metabolism and its associated diseases as well as for compound testing and personalized- and precision medicine applications.

Noninvasive Physical Plasma as Innovative and Tissue-Preserving Therapy for Women Positive for Cervical Intraepithelial Neoplasia.

(1) Background: Cervical intraepithelial neoplasia (CIN) of long-term persistence or associated with individual treatment indications often requires highly invasive treatments. These are associated with risks of bleeding, infertility, and pregnancy complications. For low- and middle-income countries (LMICs), standard treatment procedures are difficult to implement and manage. We characterized the application of the highly energized gas "noninvasive physical plasma" (NIPP) for tissue devitalization and the treatment of CIN. (2) Methods: We report the establishment of a promising tissue devitalization procedure by NIPP application. The procedure was characterized at the in vitro, ex vivo and in vivo levels. We performed the first prospective, single-armed phase-IIb trial in 20 CIN1/2 patients (NCT03218436). (3) Results: NIPP-treated cervical cancer cells used as dysplastic in vitro model exhibited significant cell growth retardation due to DNA damage, cell cycle arrest and apoptosis. Ex vivo and in vivo tissue assessments showed a highly noninvasive and tissue-preserving treatment procedure which induces transmucosal tissue devitalization. Twenty participants were treated with NIPP and attended a 24-week follow-up. Treatment success was achieved in 19 (95%) participants without postinterventional complications other than mild to moderate discomfort during application. (4) Conclusions: The results from this study preliminarily suggest that NIPP could be used for an effective and tissue-preserving treatment for CIN without the disadvantages of standard treatments. However, randomized controlled trials must confirm the efficacy and noninferiority of NIPP compared to standard treatments.

Studying metabolism with multi-organ chips: new tools for disease modelling, pharmacokinetics and pharmacodynamics.

Non-clinical models to study metabolism including animal models and cell assays are often limited in terms of species translatability and predictability of human biology. This field urgently requires a push towards more physiologically accurate recapitulations of drug interactions and disease progression in the body. Organ-on-chip systems, specifically multi-organ chips (MOCs), are an emerging technology that is well suited to providing a species-specific platform to study the various types of metabolism (glucose, lipid, protein and drug) by recreating organ-level function. This review provides a resource for scientists aiming to study human metabolism by providing an overview of MOCs recapitulating aspects of metabolism, by addressing the technical aspects of MOC development and by providing guidelines for correlation with in silico models. The current state and challenges are presented for two application areas: (i) disease modelling and (ii) pharmacokinetics/pharmacodynamics. Additionally, the guidelines to integrate the MOC data into in silico models could strengthen the predictive power of the technology. Finally, the translational aspects of metabolizing MOCs are addressed, including adoption for personalized medicine and prospects for the clinic. Predictive MOCs could enable a significantly reduced dependence on animal models and open doors towards economical non-clinical testing and understanding of disease mechanisms.

Human immunocompetent choroid-on-chip: a novel tool for studying ocular effects of biological drugs.

Disorders of the eye leading to visual impairment are a major issue that affects millions of people. On the other side ocular toxicities were described for e.g. molecularly targeted therapies in oncology and may hamper their development. Current ocular model systems feature a number of limitations affecting human-relevance and availability. To find new options for pharmacological treatment and assess mechanisms of toxicity, hence, novel complex model systems that are human-relevant and readily available are urgently required. Here, we report the development of a human immunocompetent Choroid-on-Chip (CoC), a human cell-based in vitro model of the choroid layer of the eye integrating melanocytes and microvascular endothelial cells, covered by a layer of retinal pigmented epithelial cells. Immunocompetence is achieved by perfusion of peripheral immune cells. We demonstrate controlled immune cell recruitment into the stromal compartments through a vascular monolayer and in vivo-like cytokine release profiles. To investigate applicability for both efficacy testing of immunosuppressive compounds as well as safety profiling of immunoactivating antibodies, we exposed the CoCs to cyclosporine and tested CD3 bispecific antibodies.

Development of a bi-layered cryogenic electrospun polylactic acid scaffold to study calcific aortic valve disease in a 3D co-culture model.

Calcified aortic valve disease (CAVD) is the most prevalent valve disease in the elderly. Targeted pharmacological therapies are limited since the underlying mechanisms of CAVD are not well understood. Appropriate 3D in vitro models could potentially improve our knowledge of the disease. Here, we developed a 3D in vitro aortic heart valve model that resembles the morphology of the valvular extracellular matrix and mimics the mechanical and physiological behavior of the native aortic valve fibrosa and spongiosa. We employed cryogenic electrospinning to engineer a bi-layered cryogenic electrospun scaffold (BCES) with defined morphologies that allowed valvular endothelial cell (VEC) adherence and valvular interstitial cell (VIC) ingrowth into the scaffold. Using a self-designed cell culture insert allowed us to establish the valvular co-culture simultaneously by seeding VICs on one side and VECs on the other side of the electrospun scaffold. Proof-of-principle calcification studies were successfully performed using an established osteogenic culture protocol and the here designed 3D in vitro aortic heart valve model. STATEMENT OF SIGNIFICANCE: Three-dimensional (3D) electrospun scaffolds are widely used for soft tissue engineering since they mimic the morphology of the native extracellular matrix. Several studies have shown that cells behave more naturally on 3D materials than on the commonly used stiff two-dimensional (2D) cell culture substrates, which have no biological properties. As appropriate 3D models for the study of aortic valve diseases are limited, we developed a novel bi-layered 3D in vitro test system by using the versatile technique of cryogenic electrospinning in combination with the influence of different solvents to mimic the morphology, mechanical, and cellular distribution of a native aortic heart valve leaflet. This 3D in vitro model can be used to study valve biology and heart valve-impacting diseases such as calcification to elucidate therapeutic targets.

Isolation, Integration, and Culture of Human Mature Adipocytes Leveraging Organ-on-Chip Technology.

Research on white adipose tissue (WAT), which constitutes one-fifth to one-half of the total body mass of a human's body, has gained more and more interest and attention in the era of "diabesity". In vitro research on mature human WAT is hampered by many challenges and, hence, a majority of WAT-related research is conducted using animal models as well as clinical observations and genome-wide association studies (GWAS), both featuring limitations in terms of translatability and potential for experimental interventions, respectively. Here, we describe methods to isolate primary mature human adipocytes from biopsies and to fabricate tailored organ-on-chip platforms for the long-term culture of WAT constructs.

Peristaltic on-chip pump for tunable media circulation and whole blood perfusion in PDMS-free organ-on-chip and Organ-Disc systems.

Organ-on-chip (OoC) systems have become a promising tool for personalized medicine and drug development with advantages over conventional animal models and cell assays. However, the utility of OoCs in industrial settings is still limited, as external pumps and tubing for on-chip fluid transport are dependent on error-prone, manual handling. Here, we present an on-chip pump for OoC and Organ-Disc systems, to perfuse media without external pumps or tubing. Peristaltic pumping is implemented through periodic compression of a flexible pump layer. The disc-shaped, microfluidic module contains four independent systems, each lined with endothelial cells cultured under defined, peristaltic perfusion. Both cell viability and functionality were maintained over several days shown by supernatant analysis and immunostaining. Integrated, on-disc perfusion was further used for cytokine-induced cell activation with physiologic cell responses and for whole blood perfusion assays, both demonstrating the versatility of our system for OoC applications.

Challenging the pipeline.

This commentary presents a thought experiment seeking to answer the key question: "If you were to put aside all the traditional drug discovery processes and start a new drug discovery program that places the highest priority on human and disease-relevant models throughout the entire process, how could it be done?"

Human stem cell-based retina on chip as new translational model for validation of AAV retinal gene therapy vectors.

Gene therapies using adeno-associated viruses (AAVs) are among the most promising strategies to treat or even cure hereditary and acquired retinal diseases. However, the development of new efficient AAV vectors is slow and costly, largely because of the lack of suitable non-clinical models. By faithfully recreating structure and function of human tissues, human induced pluripotent stem cell (iPSC)-derived retinal organoids could become an essential part of the test cascade addressing translational aspects. Organ-on-chip (OoC) technology further provides the capability to recapitulate microphysiological tissue environments as well as a precise control over structural and temporal parameters. By employing our recently developed retina on chip that merges organoid and OoC technology, we analyzed the efficacy, kinetics, and cell tropism of seven first- and second-generation AAV vectors. The presented data demonstrate the potential of iPSC-based OoC models as the next generation of screening platforms for future gene therapeutic studies.