A microfluidic impedance-based extended infectivity assay: combining retroviral amplification and cytopathic effect monitoring on a single lab-on-a-chip platform.

Detection, quantification and monitoring of virus - host cell interactions are of great importance when evaluating the safety of pharmaceutical products. With the wide usage of viral based vector systems in combination with mammalian cell lines for the production of biopharmaceuticals, the presence of replication competent viral particles needs to be avoided and potential hazards carefully assessed. Consequently, regulatory agencies recommend viral clearance studies using plaque assays or TCID50 assays to evaluate the efficiency of the production process in removing viruses. While plaque assays provide reliable information on the presence of viral contaminations, they are still tedious to perform and can take up to two weeks to finish. To overcome some of these limitations, we have automated, miniaturized and integrated the dual cell culture bioassay into a common lab-on-a-chip platform containing embedded electrical sensor arrays to enrich and detect infectious viruses. Results of our microfluidic single step assay show that a significant reduction in assay time down to 3 to 4 days can be achieved using simultaneous cell-based viral amplification, release and detection of cytopathic effects in a target cell line. We further demonstrate the enhancing effect of continuous fluid flow on infection of PG-4 reporter cells by newly formed and highly active virions by M. dunni cells, thus pointing to the importance of physical relevant viral-cell interactions.

Latest Trends in Biosensing for Microphysiological Organs-on-a-Chip and Body-on-a-Chip Systems.

Organs-on-chips are considered next generation in vitro tools capable of recreating in vivo like, physiological-relevant microenvironments needed to cultivate 3D tissue-engineered constructs (e.g., hydrogel-based organoids and spheroids) as well as tissue barriers. These microphysiological systems are ideally suited to (a) reduce animal testing by generating human organ models, (b) facilitate drug development and (c) perform personalized medicine by integrating patient-derived cells and patient-derived induced pluripotent stem cells (iPSCs) into microfluidic devices. An important aspect of any diagnostic device and cell analysis platform, however, is the integration and application of a variety of sensing strategies to provide reliable, high-content information on the health status of the in vitro model of choice. To overcome the analytical limitations of organs-on-a-chip systems a variety of biosensors have been integrated to provide continuous data on organ-specific reactions and dynamic tissue responses. Here, we review the latest trends in biosensors fit for monitoring human physiology in organs-on-a-chip systems including optical and electrochemical biosensors.

Optimized alamarBlue assay protocol for drug dose-response determination of 3D tumor spheroids.

The assessment of drug-dose responses is vital for the prediction of unwanted toxicological effects in modern medicine. Three-dimensional (3D) cell cultures techniques can provide in vivo-like spheroids and microtissues that resemble natural tumor function. However, formation of necrotic core and diffusion limitation of chemical compounds within these models can reduce the reproducibility and precision of standard bioassay protocols used to test two-dimensional (2D) cell cultures. Nonetheless, the accurate prediction of detrimental effects of test compounds based on functional bioassays is essential for the development of new efficient therapeutic strategies. For instance, alamarBlue® is a widely-used commercially available redox indicator dye that can evaluate metabolic activity and cellular health status in a single-step procedure however, suitability and optimization of this bioassay must be determined for each individual application scenario. Here, we optimized the standard alamarBlue® proliferation/viability protocol for tumor spheroid cultures to enhance assay precision during toxicological drug screening. We optimized the original protocol of alamarBlue® assay that usually suggests an incubation time of 2-4 hours. The key modifications of the protocol for spheroid cultures are as follows: •Aspiration of cell culture medium before drug exposure.•Replacement of drug-supplemented medium with 10% (v/v) alamarBlue® reagent mixed with culture medium.•Increase of incubation period to 24 h at 37 °C protected from light.

Combinatorial in Vitro and in Silico Approach To Describe Shear-Force Dependent Uptake of Nanoparticles in Microfluidic Vascular Models.

In the present work, we combine experimental and computational methods to define the critical shear stress as an alternative parameter for nanotoxicological and nanomedical evaluations using an in vitro microfluidic vascular model. We demonstrate that our complementary in vitro and in silico approach is well suited to assess the fluid flow velocity above which clathrin-mediated (active) nanoparticle uptake per cell decreases drastically although higher numbers of nanoparticles per cell are introduced. Results of our study revealed a critical shear stress of 1.8 dyn/cm2, where maximum active cellular nanoparticle uptake took place, followed by a 70% decrease in uptake of 249 nm nanoparticles at 10 dyn/cm2, respectively. The observed nonlinear relationship between flow velocity and nanoparticle uptake strongly suggests that fluid mechanical forces also need to be considered in order to predict potential in vivo distribution, bioaccumulation, and clearance of nanomaterials and novel nanodrugs.

Fabrication of biomimetic placental barrier structures within a microfluidic device utilizing two-photon polymerization.

The placenta is a transient organ, essential for development and survival of the unborn fetus. It interfaces the body of the pregnant woman with the unborn child and secures transport of endogenous and exogenous substances. Maternal and fetal blood are thereby separated at any time, by the so-called placental barrier. Current in vitro approaches fail to model this multifaceted structure, therefore research in the field of placental biology is particularly challenging. The present study aimed at establishing a novel model, simulating placental transport and its implications on development, in a versatile but reproducible way. The basal membrane was replicated using a gelatin-based material, closely mimicking the composition and properties of the natural extracellular matrix. The microstructure was produced by using a high-resolution 3D printing method - the two-photon polymerization (2PP). In order to structure gelatin by 2PP, its primary amines and carboxylic acids are modified with methacrylamides and methacrylates (GelMOD-AEMA), respectively. High-resolution structures in the range of a few micrometers were produced within the intersection of a customized microfluidic device, separating the x-shaped chamber into two isolated cell culture compartments. Human umbilical-vein endothelial cells (HUVEC) seeded on one side of this membrane simulate the fetal compartment while human choriocarcinoma cells, isolated from placental tissue (BeWo B30) mimic the maternal syncytium. This barrier model in combination with native flow profiles can be used to mimic the microenvironment of the placenta, investigating different pharmaceutical, clinical and biological scenarios. As proof-of-principle, this bioengineered placental barrier was used for the investigation of transcellular transport processes. While high molecular weight substances did not permeate, smaller molecules in the size of glucose were able to diffuse through the barrier in a time-depended manner. We envision to apply this bioengineered placental barrier for pathophysiological research, where altered nutrient transport is associated with health risks for the fetus.