Human In Vitro Models of Neuroenergetics and Neurometabolic Disturbances: Current Advances and Clinical Perspectives.

Neurological conditions conquer the world; they are the leading cause of disability and the second leading cause of death worldwide, and they appear all around the world in every age group, gender, nationality, and socioeconomic class. Despite the growing evidence of an immense impact of perturbations in neuroenergetics on overall brain function, only little is known about the underlying mechanisms. Especially human insights are sparse, owing to a shortage of physiologically relevant model systems. With this perspective, we aim to explore the key steps and considerations involved in developing an advanced human in vitro model for studying neuroenergetics. We discuss biological and technological strategies to meet the requirements of a predictive model, aiming at providing a guide and inspiration for future in vitro models of neuroenergetics.

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.

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.

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.

The Inflammatory Profile of Obesity and the Role on Pulmonary Bacterial and Viral Infections.

Obesity is a globally increasing health problem, entailing diverse comorbidities such as infectious diseases. An obese weight status has marked effects on lung function that can be attributed to mechanical dysfunctions. Moreover, the alterations of adipocyte-derived signal mediators strongly influence the regulation of inflammation, resulting in chronic low-grade inflammation. Our review summarizes the known effects regarding pulmonary bacterial and viral infections. For this, we discuss model systems that allow mechanistic investigation of the interplay between obesity and lung infections. Overall, obesity gives rise to a higher susceptibility to infectious pathogens, but the pathogenetic process is not clearly defined. Whereas, viral infections often show a more severe course in obese patients, the same patients seem to have a survival benefit during bacterial infections. In particular, we summarize the main mechanical impairments in the pulmonary tract caused by obesity. Moreover, we outline the main secretory changes within the expanded adipose tissue mass, resulting in chronic low-grade inflammation. Finally, we connect these altered host factors to the influence of obesity on the development of lung infection by summarizing observations from clinical and experimental data.

Integration of Electrospun Membranes into Low-Absorption Thermoplastic Organ-on-Chip.

In recent years, organ-on-chip (OoC) systems have provoked increasing interest among researchers from different disciplines. OoCs enable the recreation of in vivo-like microenvironments and the generation of a wide range of different tissues or organs in a miniaturized way. Most commonly, OoC platforms are based on microfluidic modules made of polydimethylsiloxane (PDMS). While advantageous in terms of biocompatibility, oxygen permeability, and fast prototyping amenability, PDMS features a major limitation as it absorbs small hydrophobic molecules, including many types of test compounds, hormones, and cytokines. Another common feature of OoC systems is the integration of membranes (i) to separate different tissue compartments, (ii) to confine convective perfusion to media channels, and/or (iii) to provide mechanical support for cell monolayers. Typically, porous polymer membranes are microstructured using track-etching (e.g., polyethylene terephthalate; PET) or lithography (e.g., PDMS). Although membranes of different biomechanical properties (rigid PET to elastic PDMS) have been utilized, the membrane structure and material remain mostly artificial and do not resemble in vivo conditions (extracellular matrix). Here, we report a method for the reliable fabrication and integration of electrospun membranes in OoC modules, which are made of laser-structured poly(methyl methacrylate) (PMMA). The choice of PMMA as base material provides optical parameters and biocompatibility similar to PDMS while avoiding the absorption problem. Using electrospinning for the generation of 3D membranes, microenvironments resembling the native extracellular matrix (ECM) can be generated. We tested two different kinds of electrospun membranes and established processes for a tight integration into PMMA modules. Human (microvasculature) endothelial as well as (retinal pigment) epithelial cell layers could be successfully cultured inside the systems for up to 7 days, while being either directly exposed to (endothelial cells) or protected (epithelial cells) from the shear flow. Our novel method enables the versatile fabrication of OoC platforms that can be tailored to the native environment of tissues of interest and at the same time are applicable for the testing of compounds or chemicals without constraints.

WAT-on-a-chip integrating human mature white adipocytes for mechanistic research and pharmaceutical applications.

Obesity and its numerous adverse health consequences have taken on global, pandemic proportions. White adipose tissue (WAT) - a key contributor in many metabolic diseases - contributes about one fourth of a healthy human's body mass. Despite its significance, many WAT-related pathophysiogical mechanisms in humans are still not understood, largely due to the reliance on non-human animal models. In recent years, Organ-on-a-chip (OoC) platforms have developed into promising alternatives for animal models; these systems integrate engineered human tissues into physiological microenvironment supplied by a vasculature-like microfluidic perfusion. Here, we report the development of a novel OoC that integrates functional mature human white adipocytes. The WAT-on-a-chip is a multilayer device that features tissue chambers tailored specifically for the maintenance of 3D tissues based on human primary adipocytes, with supporting nourishment provided through perfused media channels. The platform's capability to maintain long-term viability and functionality of white adipocytes was confirmed by real-time monitoring of fatty acid uptake, by quantification of metabolite release into the effluent media as well as by an intact responsiveness to a therapeutic compound. The novel system provides a promising tool for wide-ranging applications in mechanistic research of WAT-related biology, in studying of pathophysiological mechanisms in obesity and diabetes, and in R&D of pharmaceutical industry.

Merging organoid and organ-on-a-chip technology to generate complex multi-layer tissue models in a human retina-on-a-chip platform.

The devastating effects and incurable nature of hereditary and sporadic retinal diseases such as Stargardt disease, age-related macular degeneration or retinitis pigmentosa urgently require the development of new therapeutic strategies. Additionally, a high prevalence of retinal toxicities is becoming more and more an issue of novel targeted therapeutic agents. Ophthalmologic drug development, to date, largely relies on animal models, which often do not provide results that are translatable to human patients. Hence, the establishment of sophisticated human tissue-based in vitro models is of upmost importance. The discovery of self-forming retinal organoids (ROs) derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) is a promising approach to model the complex stratified retinal tissue. Yet, ROs lack vascularization and cannot recapitulate the important physiological interactions of matured photoreceptors and the retinal pigment epithelium (RPE). In this study, we present the retina-on-a-chip (RoC), a novel microphysiological model of the human retina integrating more than seven different essential retinal cell types derived from hiPSCs. It provides vasculature-like perfusion and enables, for the first time, the recapitulation of the interaction of mature photoreceptor segments with RPE in vitro. We show that this interaction enhances the formation of outer segment-like structures and the establishment of in vivo-like physiological processes such as outer segment phagocytosis and calcium dynamics. In addition, we demonstrate the applicability of the RoC for drug testing, by reproducing the retinopathic side-effects of the anti-malaria drug chloroquine and the antibiotic gentamicin. The developed hiPSC-based RoC has the potential to promote drug development and provide new insights into the underlying pathology of retinal diseases.

Stem-cell based organ-on-a-chip models for diabetes research.

Diabetes mellitus (DM) ranks among the severest global health concerns of the 21st century. It encompasses a group of chronic disorders characterized by a dysregulated glucose metabolism, which arises as a consequence of progressive autoimmune destruction of pancreatic beta-cells (type 1 DM), or as a result of beta-cell dysfunction combined with systemic insulin resistance (type 2 DM). Human cohort studies have provided evidence of genetic and environmental contributions to DM; yet, these studies are mostly restricted to investigating statistical correlations between DM and certain risk factors. Mechanistic studies, on the other hand, aimed at re-creating the clinical picture of human DM in animal models. A translation to human biology is, however, often inadequate owing to significant differences between animal and human physiology, including the species-specific glucose regulation. Thus, there is an urgent need for the development of advanced human in vitro models with the potential to identify novel treatment options for DM. This review provides an overview of the technological advances in research on DM-relevant stem cells and their integration into microphysiological environments as provided by the organ-on-a-chip technology.

Organ-on-a-Chip Systems for Women's Health Applications.

Biomedical research, for a long time, has paid little attention to the influence of sex in many areas of study, ranging from molecular and cellular biology to animal models and clinical studies on human subjects. Many studies solely rely on male cells/tissues/animals/humans, although there are profound differences in male and female physiology, which can significantly impact disease mechanisms, toxicity of compounds, and efficacy of pharmaceuticals. In vitro systems have been traditionally very limited in their capacity to recapitulate female-specific physiology and anatomy such as dynamic sex-hormone levels and the complex interdependencies of female reproductive tract organs. However, the advent of microphysiological organ-on-a-chip systems, which attempt to recreate the 3D structure and function of human organs, now gives researchers the opportunity to integrate cells and tissues from a variety of individuals. Moreover, adding a dynamic flow environment allows mimicking endocrine signaling during the menstrual cycle and pregnancy, as well as providing a controlled microfluidic environment for pharmacokinetic modeling. This review gives an introduction into preclinical and clinical research on women's health and discusses where organ-on-a-chip systems are already utilized or have the potential to deliver new insights and enable entirely new types of studies.

Integration concepts for multi-organ chips: how to maintain flexibility?!

Multi-organ platforms have an enormous potential to lead to a paradigm shift in a multitude of research domains including drug development, toxicological screening, personalized medicine as well as disease modeling. Integrating multiple organ-tissues into one microfluidic circulation merges the advantages of cell lines (human genetic background) and animal models (complex physiology) and enables the creation of more in vivo-like in vitro models. In recent years, a variety of design concepts for multi-organ platforms have been introduced, categorizable into static, semistatic and flexible systems. The most promising approach seems to be flexible interconnection of single-organ platforms to application-specific multi-organ systems. This perspective elucidates the concept of 'mix-and-match' toolboxes and discusses the numerous advantages compared with static/semistatic platforms as well as remaining challenges.

Agarose-Based Substrate Modification Technique for Chemical and Physical Guiding of Neurons In Vitro.

A new low cost and highly reproducible technique is presented that provides patterned cell culture substrates. These allow for selective positioning of cells and a chemically and mechanically directed guiding of their extensions. The patterned substrates consist of structured agarose hydrogels molded from reusable silicon micro templates. These templates consist of pins arranged equidistantly in squares, connected by bars, which mold corresponding wells and channels in the nonadhesive agarose hydrogel. Subsequent slice production with a standard vibratome, comprising the described template pattern, completes substrate production. Invertebrate neurons of locusts and pond snails are used for this application as they offer the advantage over vertebrate cells as being very large and suitable for cultivation in low cell density. Their neurons adhere to and grow only on the adhesive areas not covered by the agarose. Agarose slices of 50 µm thickness placed on glass, polystyrene, or MEA surfaces position and immobilize the neurons in the wells, and the channels guide their neurite outgrowth toward neighboring wells. In addition to the application with invertebrate neurons, the technique may also provide the potential for the application of a wide range of cell types. Long-term objective is the achievement of isolated low-density neuronal networks on MEAs or different culture substrates for various network analysis applications.