Application of a Biomimetic Nanoparticle-Based Mock Virus to Determine SARS-CoV-2 Neutralizing Antibody Levels in Blood Samples Using a Lateral Flow Assay.

The presence of neutralizing antibodies against SARS-CoV-2 in blood, acquired through previous infection or vaccination, is known to prevent the (re)occurrence of outbreaks unless the virus mutates. Therefore, the measurement of neutralizing antibodies constitutes an indispensable tool in assessing an individual's and a population's immunity against SARS-CoV-2. For this reason, we have developed an innovative lateral flow assay (LFA) capable of detecting blood-derived neutralizing antibodies using a biomimetic SARS-CoV-2 mock virus system. Here, functionalized gold nanoparticles (AuNPs) featuring the trimeric spike (S) protein at its surface imitate the virus's structure and are applied to monitor the presence and efficacy of neutralizing antibodies in blood samples. The detection principle relies on the interaction between mock virus and the immobilized angiotensin-converting enzyme 2 (ACE2) receptor, which is inhibited when neutralizing antibodies are present. To further enhance the sensitivity of our competitive assay and identify low titers of neutralizing antibodies, an additional mixing pad is embedded into the device to increase the interaction time between mock virus and neutralizing antibodies. The developed LFA is benchmarked against the WHO International Standard (21/338) and demonstrated reliable quantification of neutralizing antibodies that inhibit ACE2 binding events down to a detection limit of an antibody titer of 59 IU/mL. Additional validation using whole blood and plasma samples showed reproducible results and good comparability to a laboratory-based reference test, thus highlighting its applicability for point-of-care testing.

How Organ-on-a-Chip Technology Can Assist in Studying the Role of the Glymphatic System in Neurodegenerative Diseases.

The lack of a conventional lymphatic system that permeates throughout the entire human brain has encouraged the identification and study of alternative clearance routes within the cerebrum. In 2012, the concept of the glymphatic system, a perivascular network that fluidically connects the cerebrospinal fluid to the lymphatic vessels within the meninges via the interstitium, emerged. Although its exact mode of action has not yet been fully characterized, the key underlying processes that govern solute transport and waste clearance have been identified. This review briefly describes the perivascular glial-dependent clearance system and elucidates its fundamental role in neurodegenerative diseases. The current knowledge of the glymphatic system is based almost exclusively on animal-based measurements, but these face certain limitations inherent to in vivo experiments. Recent advances in organ-on-a-chip technology are discussed to demonstrate the technology's ability to provide alternative human-based in vitro research models. Herein, the specific focus is on how current microfluidic-based in vitro models of the neurovascular system and neurodegenerative diseases might be employed to (i) gain a deeper understanding of the role and function of the glymphatic system and (ii) to identify new opportunities for pharmacological intervention.

Monitoring the neurotransmitter release of human midbrain organoids using a redox cycling microsensor as a novel tool for personalized Parkinson's disease modelling and drug screening.

In this study, we have aimed at developing a novel electrochemical sensing approach capable of detecting dopamine, the main biomarker in Parkinson's disease, within the highly complex cell culture matrix of human midbrain organoids in a non-invasive and label-free manner. With its ability to generate organotypic structures in vitro, induced pluripotent stem cell technology has provided the basis for the development of advanced patient-derived disease models. These include models of the human midbrain, the affected region in the neurodegenerative disorder Parkinson's disease. Up to now, however, the analysis of so-called human midbrain organoids has relied on time-consuming and invasive strategies, incapable of monitoring organoid development. Using a redox-cycling approach in combination with a 3-mercaptopropionic acid self-assembled monolayer modification enabled the increase of sensor selectivity and sensitivity towards dopamine, while simultaneously reducing matrix-mediated interferences. In this work, we demonstrate the ability to detect and monitor even small differences in dopamine release between healthy and Parkinson`s disease-specific midbrain organoids over prolonged cultivation periods, which was additionally verified using liquid chromatography-multiple reaction monitoring mass spectrometry. Furthermore, the detection of a phenotypic rescue in midbrain organoids carrying a pathogenic mutation in leucine-rich repeat kinase 2, upon treatment with the leucine-rich repeat kinase 2 inhibitor II underlines the practical implementability of our sensing approach for drug screening applications as well as personalized disease modelling.

Application of a Parallelizable Perfusion Bioreactor for Physiologic 3D Cell Culture.

It is crucial but challenging to keep physiologic conditions during the cultivation of 3D cell scaffold constructs for the optimization of 3D cell culture processes. Therefore, we demonstrate the benefits of a recently developed miniaturized perfusion bioreactor together with a specialized incubator system that allows for the cultivation of multiple samples while screening different conditions. Hence, a decellularized bone matrix was tested towards its suitability for 3D osteogenic differentiation under flow perfusion conditions. Subsequently, physiologic shear stress and hydrostatic pressure (HP) conditions were optimized for osteogenic differentiation of human mesenchymal stem cells (MSCs). X-ray computed microtomography and scanning electron microscopy (SEM) revealed a closed cell layer covering the entire matrix. Osteogenic differentiation assessed by alkaline phosphatase activity and SEM was found to be increased in all dynamic conditions. Furthermore, screening of different fluid shear stress (FSS) conditions revealed 1.5 mL/min (equivalent to ∼10 mPa shear stress) to be optimal. However, no distinct effect of HP compared to flow perfusion without HP on osteogenic differentiation was observed. Notably, throughout all experiments, cells cultivated under FSS or HP conditions displayed increased osteogenic differentiation, which underlines the importance of physiologic conditions. In conclusion, the bioreactor system was used for biomaterial testing and to develop and optimize a 3D cell culture process for the osteogenic differentiation of MSCs. Due to its versatility and higher throughput efficiency, we hypothesize that this bioreactor/incubator system will advance the development and optimization of a variety of 3D cell culture processes.

Challenges and Future Perspectives in Modeling Neurodegenerative Diseases Using Organ-on-a-Chip Technology.

Neurodegenerative diseases (NDDs) affect more than 50 million people worldwide, posing a significant global health challenge as well as a high socioeconomic burden. With aging constituting one of the main risk factors for some NDDs such as Alzheimer's disease (AD) and Parkinson's disease (PD), this societal toll is expected to rise considering the predicted increase in the aging population as well as the limited progress in the development of effective therapeutics. To address the high failure rates in clinical trials, legislative changes permitting the use of alternatives to traditional pre-clinical in vivo models are implemented. In this regard, microphysiological systems (MPS) such as organ-on-a-chip (OoC) platforms constitute a promising tool, due to their ability to mimic complex and human-specific tissue niches in vitro. This review summarizes the current progress in modeling NDDs using OoC technology and discusses five critical aspects still insufficiently addressed in OoC models to date. Taking these aspects into consideration in the future MPS will advance the modeling of NDDs in vitro and increase their translational value in the clinical setting.

Sensor-integrated brain-on-a-chip platforms: Improving the predictive validity in neurodegenerative research.

Affecting millions of individuals worldwide, neurodegenerative diseases (NDDs) pose a significant and growing health concern in people over the age of 60 years. Contributing to this trend are the steady increase in the aging population coupled with a persistent lack of disease-altering treatment strategies targeting NDDs. The absence of efficient therapeutics can be attributed to high failure rates in clinical trials and the ineptness of animal models in preceding preclinical studies. To that end, in recent years, significant research effort has been dedicated to the development of human cell-based preclinical disease models characterized by a higher degree of predictive validity. However, a key requirement of any in vitro model constitutes the precise knowledge and replication of the target tissues' (patho-)physiological microenvironment. Herein, microphysiological systems have demonstrated superiority over conventional static 2D/3D in vitro cell culture systems, as they allow for the emulation and continuous monitoring of the onset, progression, and remission of disease-associated phenotypes. This review provides an overview of recent advances in the field of NDD research using organ-on-a-chip platforms. Specific focus is directed toward non-invasive sensing strategies encompassing electrical, electrochemical, and optical sensors. Additionally, promising on- and integrable off-chip sensing strategies targeting key analytes in NDDs will be presented and discussed in detail.

Archaeal ether lipids improve internalization and transfection with mRNA lipid nanoparticles.

Neutral and positively charged archaeal ether lipids (AEL) have been studied for their utilization as novel delivery systems for pDNA, showing efficient immune response with a strong memory effect while lacking noticeable toxicity. Recent technological advances placed mRNA lipid nanoparticles (LNPs) at the forefront of next-generation delivery systems; however, no study has examined AELs in mRNA delivery yet. In this study, we investigated either a crude lipid extract or the purified tetraether lipid caldarchaeol from Sulfolobus acidocaldarius as potential novel excipients for mRNA LNPs. Depending on their molar share in the respective LNP, particle uptake, and mRNA expression levels could be increased by up to 10-fold in in vitro transfection experiments using both primary cell sources (HSMM) and established cell lines (Caco-2, C2C12) compared to a well-known reference formulation. This increased efficiency might be linked to a substantial effect on endosomal escape, indicating fusogenic and lyotropic features of AELs. This study shows the high value of archaeal ether lipids for mRNA delivery and provides a solid foundation for future in vivo experiments and further research.

Engineered 3D human neurovascular model of Alzheimer's disease to study vascular dysfunction.

The blood-brain barrier (BBB) serves as a selective filter that prevents harmful substances from entering the healthy brain. Dysfunction of this barrier is implicated in several neurological diseases. In the context of Alzheimer's disease (AD), BBB breakdown plays a significant role in both the initiation and progression of the disease. This study introduces a three-dimensional (3D) self-assembled in vitro model of the human neurovascular unit to recapitulate some of the complex interactions between the BBB and AD pathologies. It incorporates primary human brain endothelial cells, pericytes and astrocytes, and stem cell-derived neurons and astrocytes harboring Familial AD (FAD) mutations. Over an extended co-culture period, the model demonstrates increased BBB permeability, dysregulation of key endothelial and pericyte markers, and morphological alterations mirroring AD pathologies. The model enables visualization of amyloid-beta (Aß) accumulation in both neuronal and vascular compartments. This model may serve as a versatile tool for neuroscience research and drug development to provide insights into the dynamic relationship between vascular dysfunction and AD pathogenesis.

Optical glucose sensor for microfluidic cell culture systems.

Glucose is the primary energy source of human cells. Therefore, monitoring glucose inside microphysiological systems (MPS) provides valuable information on the viability and metabolic state of the cultured cells. However, continuous glucose monitoring inside MPS is challenging due to a lack of suitable miniaturized sensors. Here we present an enzymatic, optical glucose sensor element for measurement inside microfluidic systems. The miniaturized glucose sensor (Ø 1 mm) is fabricated together with a reference oxygen sensor onto biocompatible, pressure-sensitive adhesive tape for easy integration inside microfluidic systems. Furthermore, the proposed microfluidic system can be used as plug and play sensor system with existing MPS. It was characterized under cell culture conditions (37 °C and pH 7.4) for five days, exhibiting minor drift (3% day-1). The influence of further cell culture parameters like oxygen concentration, pH, flow rate, and sterilization methods was investigated. The plug-and-play system was used for at-line measurements of glucose levels in (static) cell culture and achieved good agreement with a commercially available glucose sensor. In conclusion, we developed an optical glucose sensor element that can be easily integrated in microfluidic systems and is able to perform stable glucose measurements under cell culture conditions.

Accelerating the in vitro emulation of Alzheimer's disease-associated phenotypes using a novel 3D blood-brain barrier neurosphere co-culture model.

High failure rates in clinical trials for neurodegenerative disorders such as Alzheimer's disease have been linked to an insufficient predictive validity of current animal-based disease models. This has created an increasing demand for alternative, human-based models capable of emulating key pathological phenotypes in vitro. Here, a three-dimensional Alzheimer's disease model was developed using a compartmentalized microfluidic device that combines a self-assembled microvascular network of the human blood-brain barrier with neurospheres derived from Alzheimer's disease-specific neural progenitor cells. To shorten microfluidic co-culture times, neurospheres were pre-differentiated for 21 days to express Alzheimer's disease-specific pathological phenotypes prior to the introduction into the microfluidic device. In agreement with post-mortem studies and Alzheimer's disease in vivo models, after 7 days of co-culture with pre-differentiated Alzheimer's disease-specific neurospheres, the three-dimensional blood-brain barrier network exhibited significant changes in barrier permeability and morphology. Furthermore, vascular networks in co-culture with Alzheimer's disease-specific microtissues displayed localized ß-amyloid deposition. Thus, by interconnecting a microvascular network of the blood-brain barrier with pre-differentiated neurospheres the presented model holds immense potential for replicating key neurovascular phenotypes of neurodegenerative disorders in vitro.

Screening for Best Neuronal-Glial Differentiation Protocols of Neuralizing Agents Using a Multi-Sized Microfluidic Embryoid Body Array.

Stem cell technology and embryonic stem cell models are of great interest in biomedical research since they provide deeper insights into, e.g., neurogenesis and early mammalian brain development. Despite their great scientific potential, the reliable establishment of three-dimensional embryoid bodies (EBs) remains a major challenge, and the current lack of standardization and comparability is still limiting a broader application and translation of stem cell technology. Among others, a vital aspect for the reliable formation of EBs is optimizing differentiation protocols since organized differentiation is influenced by soluble inducers and EB size. A microfluidic biochip array was employed to automate cell loading and optimize directed neuronal and astrocytic differentiation protocols using murine P19 embryoid bodies to facilitate reliable embryonic stem cell differentiation. Our gravity-driven microfluidic size-controlled embryoid body-on-a-chip system allows (a) the robust operation and cultivation of up to 90 EBs in parallel and (b) the reproducible generation of five increasing sizes ranging from 300 µm to 1000 µm diameters. A comparative study adds two differentiation-inducers such as retinoic acid and EC23 to size-controlled embryoid bodies to identify the optimal differentiation protocol. Our study revealed a 1.4 to 1.9-fold higher neuron and astrocyte expression in larger embryoid bodies (above 750 µm) over smaller-sized EBs (below 450 µm), thus highlighting the importance of EB size in the establishment of robust neurodevelopmental in vitro models.

Recent Advances in Additive Manufacturing and 3D Bioprinting for Organs-On-A-Chip and Microphysiological Systems.

The re-creation of physiological cellular microenvironments that truly resemble complex in vivo architectures is the key aspect in the development of advanced in vitro organotypic tissue constructs. Among others, organ-on-a-chip technology has been increasingly used in recent years to create improved models for organs and tissues in human health and disease, because of its ability to provide spatio-temporal control over soluble cues, biophysical signals and biomechanical forces necessary to maintain proper organotypic functions. While media supply and waste removal are controlled by microfluidic channel by a network the formation of tissue-like architectures in designated micro-structured hydrogel compartments is commonly achieved by cellular self-assembly and intrinsic biological reorganization mechanisms. The recent combination of organ-on-a-chip technology with three-dimensional (3D) bioprinting and additive manufacturing techniques allows for an unprecedented control over tissue structures with the ability to also generate anisotropic constructs as often seen in in vivo tissue architectures. This review highlights progress made in bioprinting applications for organ-on-a-chip technology, and discusses synergies and limitations between organ-on-a-chip technology and 3D bioprinting in the creation of next generation biomimetic in vitro tissue models.

Dependence of mitochondrial function on the filamentous actin cytoskeleton in cultured mesenchymal stem cells treated with cytochalasin B.

Owing to their self-renewal and multi-lineage differentiation capability, mesenchymal stem cells (MSCs) hold enormous potential in regenerative medicine. A prerequisite for a successful MSC therapy is the rigorous investigation of their function after in vitro cultivation. Damages introduced to mitochondria during cultivation adversely affect MSCs function and can determine their fate. While it has been shown that microtubules and vimentin intermediate filaments are important for mitochondrial dynamics and active mitochondrial transport within the cytoplasm of MSCs, the role of filamentous actin in this process has not been fully understood yet. To gain a deeper understanding of the interdependence between mitochondrial function and the cytoskeleton, we applied cytochalasin B to disturb the filamentous actin-based cytoskeleton of MSCs. In this study we combined conventional functional assays with a state-of-the-art oxygen sensor-integrated microfluidic device to investigate mitochondrial function. We demonstrated that cytochalasin B treatment at a dose of 16 µM led to a decrease in cell viability with high mitochondrial membrane potential, increased oxygen consumption rate, disturbed fusion and fission balance, nuclear extrusion and perinuclear accumulation of mitochondria. Treatment of MSCs for 48 h ultimately led to nuclear fragmentation, and activation of the intrinsic pathway of apoptotic cell death. Importantly, we could show that mitochondrial function of MSCs can efficiently recover from the damage to the filamentous actin-based cytoskeleton over a period of 24 h. As a result of our study, a causative connection between the filamentous actin-based cytoskeleton and mitochondrial dynamics was demonstrated.

Breaking the Third Wall: Implementing 3D-Printing Technics to Expand the Complexity and Abilities of Multi-Organ-on-a-Chip Devices.

The understanding that systemic context and tissue crosstalk are essential keys for bridging the gap between in vitro models and in vivo conditions led to a growing effort in the last decade to develop advanced multi-organ-on-a-chip devices. However, many of the proposed devices have failed to implement the means to allow for conditions tailored to each organ individually, a crucial aspect in cell functionality. Here, we present two 3D-print-based fabrication methods for a generic multi-organ-on-a-chip device: One with a PDMS microfluidic core unit and one based on 3D-printed units. The device was designed for culturing different tissues in separate compartments by integrating individual pairs of inlets and outlets, thus enabling tissue-specific perfusion rates that facilitate the generation of individual tissue-adapted perfusion profiles. The device allowed tissue crosstalk using microchannel configuration and permeable membranes used as barriers between individual cell culture compartments. Computational fluid dynamics (CFD) simulation confirmed the capability to generate significant differences in shear stress between the two individual culture compartments, each with a selective shear force. In addition, we provide preliminary findings that indicate the feasibility for biological compatibility for cell culture and long-term incubation in 3D-printed wells. Finally, we offer a cost-effective, accessible protocol enabling the design and fabrication of advanced multi-organ-on-a-chip devices.

A Decade of Organs-on-a-Chip Emulating Human Physiology at the Microscale: A Critical Status Report on Progress in Toxicology and Pharmacology.

Organ-on-a-chip technology has the potential to accelerate pharmaceutical drug development, improve the clinical translation of basic research, and provide personalized intervention strategies. In the last decade, big pharma has engaged in many academic research cooperations to develop organ-on-a-chip systems for future drug discoveries. Although most organ-on-a-chip systems present proof-of-concept studies, miniaturized organ systems still need to demonstrate translational relevance and predictive power in clinical and pharmaceutical settings. This review explores whether microfluidic technology succeeded in paving the way for developing physiologically relevant human in vitro models for pharmacology and toxicology in biomedical research within the last decade. Individual organ-on-a-chip systems are discussed, focusing on relevant applications and highlighting their ability to tackle current challenges in pharmacological research.

Bridging the academic-industrial gap: application of an oxygen and pH sensor-integrated lab-on-a-chip in nanotoxicology.

Translation of advanced cell-based assays exhibiting a higher degree of automation, miniaturization, and integration of complementary sensing functions is mainly limited by the development of industrial-relevant prototypes that can be readily produced in larger volumes. Despite the increasing number of academic publications in recent years, the manufacturability of these microfluidic cell cultures systems is largely ignored, thus severely restricting their implementation in routine toxicological applications. We have developed a dual-sensor integrated microfluidic cell analysis platform using industrial specifications, materials, and fabrication methods to conduct risk assessment studies of engineered nanoparticles to overcome this academic-industrial gap. Non-invasive and time-resolved monitoring of cellular oxygen uptake and metabolic activity (pH) in the absence and presence of nanoparticle exposure is accomplished by integrating optical sensor spots into a cyclic olefin copolymer (COC)-based microfluidic platform. Results of our nanotoxicological study, including two physiological cell barriers that are essential in the protection from exogenous factors, the intestine (Caco-2) and the vasculature (HUVECs) showed that the assessment of the cells' total energy metabolism is ideally suited to rapidly detect cytotoxicities. Additional viability assay verification using state-of-the-art dye exclusion assays for nanotoxicology demonstrated the similarity and comparability of our results, thus highlighting the benefits of employing a compact and cost-efficient microfluidic dual-sensor platform as a pre-screening tool in nanomaterial risk assessment and as a rapid quality control measure in medium to high-throughput settings.

Establishment of a human three-dimensional chip-based chondro-synovial coculture joint model for reciprocal cross talk studies in arthritis research.

Rheumatoid arthritis is characterised by a progressive, intermittent inflammation at the synovial membrane, which ultimately leads to the destruction of the synovial joint. The synovial membrane as the joint capsule's inner layer is lined with fibroblast-like synoviocytes that are the key player supporting persistent arthritis leading to bone erosion and cartilage destruction. While microfluidic models that model molecular aspects of bone erosion between bone-derived cells and synoviocytes have been established, RA's synovial-chondral axis has not yet been realised using a microfluidic 3D model based on human patient in vitro cultures. Consequently, we established a chip-based three-dimensional tissue coculture model that simulates the reciprocal cross talk between individual synovial and chondral organoids. When co-cultivated with synovial organoids, we could demonstrate that chondral organoids induce a higher degree of cartilage physiology and architecture and show differential cytokine response compared to their respective monocultures highlighting the importance of reciprocal tissue-level cross talk in the modelling of arthritic diseases.

Stiffness Matters: Fine-Tuned Hydrogel Elasticity Alters Chondrogenic Redifferentiation.

Biomechanical cues such as shear stress, stretching, compression, and matrix elasticity are vital in the establishment of next generation physiological in vitro tissue models. Matrix elasticity, for instance, is known to guide stem cell differentiation, influence healing processes and modulate extracellular matrix (ECM) deposition needed for tissue development and maintenance. To better understand the biomechanical effect of matrix elasticity on the formation of articular cartilage analogs in vitro, this study aims at assessing the redifferentiation capacity of primary human chondrocytes in three different hydrogel matrices of predefined matrix elasticities. The hydrogel elasticities were chosen to represent a broad spectrum of tissue stiffness ranging from very soft tissues with a Young's modulus of 1 kPa up to elasticities of 30 kPa, representative of the perichondral-space. In addition, the interplay of matrix elasticity and transforming growth factor beta-3 (TGF-ß3) on the redifferentiation of primary human articular chondrocytes was studied by analyzing both qualitative (viability, morphology, histology) and quantitative (RT-qPCR, sGAG, DNA) parameters, crucial to the chondrotypic phenotype. Results show that fibrin hydrogels of 30 kPa Young's modulus best guide chondrocyte redifferentiation resulting in a native-like morphology as well as induces the synthesis of physiologic ECM constituents such as glycosaminoglycans (sGAG) and collagen type II. This comprehensive study sheds light onto the mechanobiological impact of matrix elasticity on formation and maintenance of articular cartilage and thus represents a major step toward meeting the need for advanced in vitro tissue models to study both re- and degeneration of articular cartilage.

FTIR spectroscopy as a novel analytical approach for investigation of glucose transport and glucose transport inhibition studies in transwell in vitro barrier models.

Glucose transport is key for cellular metabolism as well as physiological function and is maintained via passive facilitated and active sodium-glucose linked transport routes. Here, we present for the first time Fourier-transform infrared spectroscopy as a novel approach for quantification of apical-to-basolateral glucose transport of in vitro cell barrier models using liver, lung, intestinal and placental cancer cell lines. Results of our comparative study revealed that distinct differences could be observed upon subjection to transport inhibitors.

The Usual Suspects 2019: of Chips, Droplets, Synthesis, and Artificial Cells.

Synthetic biology aims to understand fundamental biological processes in more detail than possible for actual living cells. Synthetic biology can combat decomposition and build-up of artificial experimental models under precisely controlled and defined environmental and biochemical conditions. Microfluidic systems can provide the tools to improve and refine existing synthetic systems because they allow control and manipulation of liquids on a micro- and nanoscale. In addition, chip-based approaches are predisposed for synthetic biology applications since they present an opportune technological toolkit capable of fully automated high throughput and content screening under low reagent consumption. This review critically highlights the latest updates in microfluidic cell-free and cell-based protein synthesis as well as the progress on chip-based artificial cells. Even though progress is slow for microfluidic synthetic biology, microfluidic systems are valuable tools for synthetic biology and may one day help to give answers to long asked questions of fundamental cell biology and life itself.