Search

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. [ABSTRACT FROM AUTHOR]

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: β-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. [ABSTRACT FROM AUTHOR]

Organ-on-chip (OoC) technology bridges the principles of biology and engineering to create a new generation of in vitro models and involves highly interdisciplinary collaboration across STEM disciplines. Training the next generation of scientists, technicians and policy makers is a challenge that requires a tailored effort. To promote the qualification, usability, uptake and long-term development of OoC technology, we designed a questionnaire to evaluate the key aspects for training, identify the major stakeholders to be trained, their professional level and specific skillset. The 151 respondents unanimously agreed on the need to train the next generation of OoC researchers and that the training should be provided early, in interdisciplinary subjects and throughout the researchers' career. We identified two key training priorities: (i) training scientists with a biology background in microfabrication and microfluidics principles and (ii) training OoC developers in pharmacology/toxicology. This makes training in OoC a transdisciplinary challenge rather than an interdisciplinary one. The data acquired and analyzed here serves to guide training initiatives for preparing competent and transdisciplinary researchers, capable of assuring the successful development and application of OoC technologies in academic research, pharmaceutical/chemical/cosmetic industries, personalized medicine and clinical trials on chip. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. 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. [Display omitted] [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Membranes play a crucial role in many microfluidic systems, enabling versatile applications in highly diverse research fields. However, the tight and robust integration of membranes into microfluidic systems requires complex fabrication processes. Most integration approaches, so far, rely on polydimethylsiloxane (PDMS) as base material for the microfluidic chips. Several limitations of PDMS have resulted in the transition of many microfluidic approaches to PDMS-free systems using alternative materials such as thermoplastics. To integrate membranes in those PDMS-free systems, novel alternative approaches are required. This review provides an introduction into microfluidic systems applying membrane technology for analytical systems and organ-on-chip as well as a comprehensive overview of methods for the integration of membranes into PDMS-free systems. The overview and examples will provide a valuable resource and starting point for any researcher that is aiming at implementing membranes in microfluidic systems without using PDMS. [ABSTRACT FROM AUTHOR]

Organ-on-a-chip (OoC) systems have evolved to a promising alternative to animal testing and traditional cell assays in drug development and enable personalization for precision medicine. So far, most OoCs do not fully exploit the potential of microfluidic systems regarding parallelization and automation. To date, many OoCs still consist of individual units, integrating only one single tissue per chip, and rely on manual, error-prone handling. However, with limited parallelization and automation, OoCs remain a low-throughput technology, preventing their widespread application in industry. To advance the concept of microphysiological systems and to overcome the limitations of current OoCs, we developed the Organ-on-a-disc (Organ-Disc) technology. Driven only by rotation, Organ-Discs enable the parallelized generation and culture of multiple 3D cell constructs per disc. We fabricated polydimethylsiloxane-free Organ-Discs using thermoplastic materials and scalable fabrication techniques. Utilizing precisely controllable centrifugal forces, cells were loaded simultaneously into 20 tissue chambers, where they formed uniform cell pellets. Subsequently, the cells compacted into dense 3D cell constructs and were cultured under vasculature-like perfusion through pump- and tubing-free, centrifugal pumping, solely requiring a low-speed rotation (<1 g) of the Organ-Disc. Here, we provide a proof-of-concept of the Organ-Disc technology, showing the parallelized generation of tissue-like cell constructs and demonstrating the controlled centrifugal perfusion. Furthermore, Organ-Discs enable versatile tissue engineering, generating cell constructs with a customizable shape and a layered multi-cell type structure. Overall, the Organ-Disc provides a user-friendly platform technology for the parallelization and automation of microphysiological systems, bringing this technology one-step closer to high-throughput applications in industry. [ABSTRACT FROM AUTHOR]

Pancreatic islet isolation from donor pancreases is an essential step for the transplantation of insulin‐secreting β‐cells as a therapy to treat type 1 diabetes mellitus. This process however damages islet basement membranes, which can lead to islet dysfunction or death. Posttransplantation, islets are further stressed by a hypoxic environment and immune reactions that cause poor engraftment and graft failure. The current standards to assess islet quality before transplantation are destructive procedures, performed on a small islet population that does not reflect the heterogeneity of large isolated islet batches. In this study, we incorporated fluorescence lifetime imaging microscopy (FLIM) into a pancreas‐on‐chip system to establish a protocol to noninvasively assess the viability and functionality of pancreatic β‐cells in a three‐dimensional in vitro model (= pseudo‐islets). We demonstrate how (pre‐) hypoxic β‐cell‐composed pseudo‐islets can be discriminated from healthy functional pseudo‐islets according to their FLIM‐based metabolic profiles. The use of FLIM during the pretransplantation pancreatic islet selection process has the potential to improve the outcome of β‐cell islet transplantation. [ABSTRACT FROM AUTHOR]

Soft lithography techniques using polydimethylsiloxane (PDMS) are a cornerstone of microfluidic microdevices and emerging technologies such as microphysiological systems (MPS). Most of these systems employ hydrophobic small molecules during either stem cell differentiation, drug screening, or organoid development. However, due to PDMS's structure and hydrophobicity, lipophilic molecules are strongly absorbed creating unpredictable concentrations of mitogens, drugs, differentiation factors, and analytes, which is a major limitation in its use for biological applications. In this study, several catechol‐functionalized calix[4]arene based macrocyclic polyphenols (MPPs) are synthesized and coated on PDMS through a dip‐coating or flow through process. One molecule, MPP5cone, synthesized from catechol and resorcinol in its cone isomer form, increases the hydrophilicity of PDMS and drastically reduces the absorption of a number of hydrophobic drug surrogates, while preserving high oxygen permeability, good cell viability and function. However, simple rules of molecular absorption based on Log P are not observed, suggesting screening barrier coatings for PDMS with single probes is not sufficient. The coating procedure is easily translated to microfluidic devices by infusion through channels with a pump, and therefore should find use in applications where molecular absorption into PDMS is a significant problem. [ABSTRACT FROM AUTHOR]

Organ-on-a-chip systems possess a promising future as drug screening assays and as testbeds for disease modeling in the context of both single-organ systems and multi-organ-chips. Although it comprises approximately one fourth of the body weight of a healthy human, an organ frequently overlooked in this context is white adipose tissue (WAT). WAT-on-a-chip systems are required to create safety profiles of a large number of drugs due to their interactions with adipose tissue and other organs via paracrine signals, fatty acid release, and drug levels through sequestration. We report a WAT-on-a-chip system with a footprint of less than 1 mm2 consisting of a separate media channel and WAT chamber connected via small micropores. Analogous to the in vivo blood circulation, convective transport is thereby confined to the vasculature-like structures and the tissues protected from shear stresses. Numerical and analytical modeling revealed that the flow rates in the WAT chambers are less than 1/100 of the input flow rate. Using optimized injection parameters, we were able to inject pre-adipocytes, which subsequently formed adipose tissue featuring fully functional lipid metabolism. The physiologically relevant microfluidic environment of the WAT-chip supported long term culture of the functional adipose tissue for more than two weeks. Due to its physiological, highly controlled, and computationally predictable character, the system has the potential to be a powerful tool for the study of adipose tissue associated diseases such as obesity and type 2 diabetes. [ABSTRACT FROM AUTHOR]

Human organ-on-a-chip systems for drug screening have evolved as feasible alternatives to animal models, which are unreliable, expensive, and at times erroneous. While chips featuring single organs can be of great use for both pharmaceutical testing and basic organ-level studies, the huge potential of the organ-on-a-chip technology is revealed by connecting multiple organs on one chip to create a single integrated system for sophisticated fundamental biological studies and devising therapies for disease. Furthermore, since most organ-on-a-chip systems require special protocols with organ-specific media for the differentiation and maturation of the tissues, multi-organ systems will need to be temporally customizable and flexible in terms of the time point of connection of the individual organ units. We present a customizable Lego®-like plug & play system, μOrgano, which enables initial individual culture of single organ-on-a-chip systems and subsequent connection to create integrated multi-organ microphysiological systems. As a proof of concept, the μOrgano system was used to connect multiple heart chips in series with excellent cell viability and spontaneously physiological beat rates. [ABSTRACT FROM AUTHOR]

Although adhesive interactions between cells and nanostructured interfaces have been studied extensively, there is a paucity of data on how nanostructured interfaces repel cells by directing cell migration and cell-colony organization. Here, by using multiphoton ablation lithography to pattern surfaces with nanoscale craters of various aspect ratios and pitches, we show that the surfaces altered the cells' focal-adhesion size and distribution, thus affecting cell morphology, migration and ultimately localization. We also show that nanocrater pitch can disrupt the formation of mature focal adhesions to favour the migration of cells towards higher-pitched regions, which present increased planar area for the formation of stable focal adhesions. Moreover, by designing surfaces with variable pitch but constant nanocrater dimensions, we were able to create circular and striped cellular patterns. Our surface-patterning approach, which does not involve chemical treatments and can be applied to various materials, represents a simple method to control cell behaviour on surfaces. [ABSTRACT FROM AUTHOR]