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.

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.

Tomorrow today: organ-on-a-chip advances towards clinically relevant pharmaceutical and medical in vitro models.

Organ-on-a-chip technology offers the potential to recapitulate human physiology by keeping human cells in a precisely controlled and artificial tissue-like microenvironment. The current and potential advantages of organs-on-chips over conventional cell cultures systems and animal models have captured the attention of scientists, clinicians and policymakers as well as advocacy groups in the past few years. Recent advances in tissue engineering and stem cell research are also aiding the development of clinically relevant chip-based organ and diseases models with organ level physiology for drug screening, biomedical research and personalized medicine. Here, the latest advances in organ-on-a-chip technology are reviewed and future clinical applications discussed.

Engineering of three-dimensional pre-vascular networks within fibrin hydrogel constructs by microfluidic control over reciprocal cell signaling.

Reengineering functional vascular networks in vitro remains an integral part in tissue engineering, since the incorporation of non-perfused tissues results in restricted nutrient supply and limited waste removal. Microfluidic devices are routinely used to mimic both physiological and pathological vascular microenvironments. Current procedures either involve the investigation of growth factor gradients and interstitial flow on endothelial cell sprouting alone or on the heterotypic cell-cell interactions between endothelial and mural cells. However, limited research has been conducted on the influence of flow on co-cultures of these cells. Here, we exploited the ability of microfluidics to create and monitor spatiotemporal gradients to investigate the influence of growth factor supply and elution on vascularization using static as well as indirect and direct flow setups. Co-cultures of human adipose-derived stem/stromal cells and human umbilical vein endothelial cells embedded in fibrin hydrogels were found to be severely affected by diffusion limited growth factor gradients as well as by elution of reciprocal signaling molecules during both static and flow conditions. Static cultures formed pre-vascular networks up to a depth of 4 mm into the construct with subsequent decline due to diffusion limitation. In contrast, indirect flow conditions enhanced endothelial cell sprouting but failed to form vascular networks. Additionally, complete inhibition of pre-vascular network formation was observable for direct application of flow through the hydrogel with decline of endothelial cell viability after seven days. Using finite volume CFD simulations of different sized molecules vital for pre-vascular network formation into and out of the hydrogel constructs, we found that interstitial flow enhances growth factor supply to the cells in the bulk of the chamber but elutes cellular secretome, resulting in truncated, premature vascularization.

Every Breath You Take: Non-invasive Real-Time Oxygen Biosensing in Two- and Three-Dimensional Microfluidic Cell Models.

Knowledge on the availability of dissolved oxygen inside microfluidic cell culture systems is vital for recreating physiological-relevant microenvironments and for providing reliable and reproducible measurement conditions. It is important to highlight that in vivo cells experience a diverse range of oxygen tensions depending on the resident tissue type, which can also be recreated in vitro using specialized cell culture instruments that regulate external oxygen concentrations. While cell-culture conditions can be readily adjusted using state-of-the-art incubators, the control of physiological-relevant microenvironments within the microfluidic chip, however, requires the integration of oxygen sensors. Although several sensing approaches have been reported to monitor oxygen levels in the presence of cell monolayers, oxygen demands of microfluidic three-dimensional (3D)-cell cultures and spatio-temporal variations of oxygen concentrations inside two-dimensional (2D) and 3D cell culture systems are still largely unknown. To gain a better understanding on available oxygen levels inside organ-on-a-chip systems, we have therefore developed two different microfluidic devices containing embedded sensor arrays to monitor local oxygen levels to investigate (i) oxygen consumption rates of 2D and 3D hydrogel-based cell cultures, (ii) the establishment of oxygen gradients within cell culture chambers, and (iii) influence of microfluidic material (e.g., gas tight vs. gas permeable), surface coatings, cell densities, and medium flow rate on the respiratory activities of four different cell types. We demonstrate how dynamic control of cyclic normoxic-hypoxic cell microenvironments can be readily accomplished using programmable flow profiles employing both gas-impermeable and gas-permeable microfluidic biochips.

Recent advances in microfluidic technologies for cell-to-cell interaction studies.

Microfluidic cell cultures are ideally positioned to become the next generation of in vitro diagnostic tools for biomedical research, where key biological processes such as cell signalling and dynamic cell-to-cell interactions can be reliably analysed under reproducible physiological cell culture conditions. In the last decade, a large number of microfluidic cell analysis systems have been developed for a variety of applications including drug target optimization, drug screening and toxicological testing. More recently, advanced in vitro microfluidic cell culture systems have emerged that are capable of replicating the complex three-dimensional architectures of tissues and organs and thus represent valid biological models for investigating the mechanism and function of human tissue structures, as well as studying the onset and progression of diseases such as cancer. In this review, we present the most important developments in single-cell, 2D and 3D microfluidic cell culture systems for studying cell-to-cell interactions published over the last 6 years, with a focus on cancer research and immunotherapy, vascular models and neuroscience. In addition, the current technological development of microdevices with more advanced physiological cell microenvironments that integrate multiple organ models, namely, the so-called body-, human- and multi-organ-on-a-chip, is reviewed.

Bedside Immune Monitoring: An Automated Immunoassay Platform for Quantification of Blood Biomarkers in Patient Serum within 20 Minutes.

This Article presents an automated, compact, and self-contained system for sensitive quantitative detection of blood biomarkers. A disposable microfluidic chip, prefilled with biomarker-specific reagents and magnetic beads, can be processed fully automatically by a readout platform, enabling an immunoassay-based analysis with a processing time from sample incubation to signal analysis of 20 min. Novel concepts for on-chip vortexing of the magnetic beads and on-chip reagent storage and actuation were developed. A lens-free photodiode readout system represents a cost-efficient approach for detecting the chemiluminescent signal. IL-8 spiked serum samples were measured with a high reproducibility and a limit of detection of 2.05 pg·mL-1. The system was validated with undiluted serum samples collected from trauma patients at the intensive care unit. The developed platform demonstrated good correlation with the clinical reference method, and the clinical trajectory course of IL-8 could be sufficiently followed. With an automated assay approach and an easily adaptable protocol, this portable platform has the potential to be utilized as a universal instrument for analyzing proteins in small sample volumes (<25 µL) in point-of-care settings.

A compact and integrated immunoassay with on-chip dispensing and magnetic particle handling.

We present a compact diagnostic platform for a rapid and sensitive detection of plasma biomarkers. The platform consists of a disposable microfluidic polymer chip, a processing device including a lens-free and cost efficient sensor system and a setup for dispersion of magnetic particles. The biomarkers of interest are quantified by magnetic bead based immunoassays with chemiluminescent readout technology. With a novel system for dispersion and manipulation of the magnetic particles in combination with chemiluminescence detection, the sensitivity of the immunoassay is improved and enables a rapid assay in a microfluidic format. In the disposable chip, extra chambers for storage and dispensing of biomarker specific reagents are integrated, which reduce the need of external dosing devices and thereby the cost of the platform is decreased. Plasma biomarkers for monitoring of sepsis could be quantified at 10 pg/mL concentrations within a total time of 30 min by the present system. This contribution is a fundamental step towards the development of an automatic and compact Point-of-Care testing device for monitoring of patients at the intensive care unit.