Phaseguide assisted liquid lamination for magnetic particle-based assays

We have developed a magnetic particle-based assay platform in which functionalised magnetic particles are transferred sequentially through laminated volumes of reagents and washing buffers. Lamination of aqueous liquids is achieved via the use of phaseguide technology; microstructures that control the advancing air–liquid interface of solutions as they enter a microfluidic chamber. This allows manual filling of the device, eliminating the need for external pumping systems, and preparation of the system requires only a few minutes. Here, we apply the platform to two on-chip strategies: (i) a one-step streptavidin–biotin binding assay, and (ii) a two-step C-reactive protein immunoassay. With these, we demonstrate how condensing multiple reaction and washing processes into a single step significantly reduces procedural times, with both assay procedures requiring less than 8 seconds

Design and analysis of a microplate assay in the presence of multiple restrictions on the randomization

Experiments using multi-step protocols often involve several restrictions on the randomization. For a specific application to in vitro testing on microplates, a design was required with both a split-plot and a strip-plot structure. On top of two-level treatment factors and the factors that define the randomization restrictions, a multi-level fixed blocking factor not involving further restrictions on the randomization had to be added. We develop a step-by-step approach to construct a design for the microplate experiment and analyze a response. To consolidate the approach, we study various alternative scenarios for the experiment.Comment: 31 pages, 13 tables, 4 figure

A versatile multiplexed assay to quantify intracellular ROS and cell viability in 3D on-a-chip models

Reactive oxygen species (ROS) have different properties and biological functions. They contribute to cell signaling and, in excessive amounts, to oxidative stress (OS). Although ROS is pivotal in a wide number of physiological systems and pathophysiological processes, direct quantification in vivo is quite challenging and mainly limited to in vitro studies. Even though advanced in vitro cell culture techniques, like on-a-chip culture, have overcome the lack of crucial in vivo-like physiological aspects in 2D culture, the majority of in vitro ROS quantification studies are generally performed in 2D. Here we report the development, application, and validation of a multiplexed assay to quantify ROS and cell viability in organ-on-a-chip models. The assay utilizes three dyes to stain live cells for ROS, dead cells, and DNA. Confocal images were analyzed to quantify ROS probes and determine the number of nuclei and dead cells. We found that, in contrast to what has been reported with 2D cell culture, on-a-chip models are more prone to scavenge ROS rather than accumulate them. The assay is sensitive enough to distinguish between different phenotypes of endothelial cells (ECs) based on the level of OS to detect higher level in tumor than normal cells. Our results indicate that the use of physiologically relevant models and this assay could help unravelling the mechanisms behind OS and ROS accumulation. A further step could be taken in data analysis by implementing AI in the pipeline to also analyze images for morphological changes to have an even broader view of OS mechanism

Acoustic Characterization of a Vessel-on-a-Chip Microfluidic System for Ultrasound-Mediated Drug Delivery

Ultrasound in the presence of gas-filled microbubbles can be used to enhance local uptake of drugs and genes. To study the drug delivery potential and its underlying physical and biological mechanisms, an in vitro vessel model should ideally include 3D cell culture, perfusion flow, and membranefree soft boundaries. Here, we propose an organ-on-a-chip microfluidic platform to study ultrasound-mediated drug delivery: the OrganoPlate. The acoustic propagation into the OrganoPlate was determined to assess the feasibility of controlled microbubble actuation, which is required to study the microbubble-cell interaction for drug delivery. The pressure field in the OrganoPlate was characterized non-invasively by studying experimentally the well-known response of microbubbles and by simulating the acoustic wave propagation in the system. Microbubble dynamics in the OrganoPlate were recorded with the Brandaris 128 ultrahigh speed camera (17 Mfps) and a control experiment was performed in an OptiCell, an in vitro monolayer cell culture chamber that is conventionally used to study ultrasound-mediated d

Endoglin and squamous cell carcinomas

Despite the fact that the role of endoglin on endothelial cells has been extensively described, its expression and biological role on (epithelial) cancer cells is still debatable. Especially its function on squamous cell carcinoma (SCC) cells is largely unknown. Therefore, we investigated SCC endoglin expression and function in three types of SCCs; head and neck (HNSCC), esophageal (ESCC) and vulvar (VSCC) cancers. Endoglin expression was evaluated in tumor specimens and 14 patient-derived cell lines. Next to being expressed on angiogenic endothelial cells, endoglin is selectively expressed by individual SCC cells in tumor nests. Patient derived HNSCC, ESCC and VSCC cell lines express varying levels of endoglin with high interpatient variation. To assess the function of endoglin in signaling of TGF-β ligands, endoglin was overexpressed or knocked out or the signaling was blocked using TRC105, an endoglin neutralizing antibody. The endoglin ligand BMP-9 induced strong phosphorylation of SMAD1 independent of expression of the type-I receptor ALK1. Interestingly, we observed that endoglin overexpression leads to strongly increased soluble endoglin levels, which in turn decreases BMP-9 signaling. On the functional level, endoglin, both in a ligand dependent and independent manner, did not influence proliferation or migration of the SCC cells. In conclusion, these data show endoglin expression on individual cells in the tumor nests in SCCs and a role for (soluble) endoglin in paracrine signaling, without directly affecting proliferation or migration in an autocrine manner.</p

Membrane-free culture and real-time barrier integrity assessment of perfused intestinal epithelium tubes

In vitro models that better reflect in vivo epithelial barrier (patho-)physiology are urgently required to predict adverse drug effects. Here we introduce extracellular matrix-supported intestinal tubules in perfused microfluidic devices, exhibiting tissue polarization and transporter expression. Forty leak-tight tubules are cultured in parallel on a single plate and their response to pharmacological stimuli is recorded over 125 h using automated imaging techniques. A study comprising 357 gut tubes is performed, of which 93% are leak tight before exposure. EC50-time curves could be extracted that provide insight into both concentration and exposure time response. Full compatibility with standard equipment and user-friendly operation make this Organ-on-a-Chip platform readily applicable in routine laboratories

The Effect of Various Levels of Dietary Starch on Glycogen Replenishment in the Light Working Horse

Nine Quarter Horses (2 to 7 yr; 409 to 494 kg BW) were used in a 3 x 3 Latin square with replication study lasting 105 d to determine the effect of various levels of dietary starch on glycogen replenishment in the light working horse. Horses were fed 1 % BW/d in Coastal Bermudagrass hay with remaining calories met by a high starch (HS), medium starch (MS), or low starch (LS) concentrate. After a 7 d washout period, horses were transitioned to 1 of the 3 diets over 7 d for a 14 d treatment period where they were then worked to fatigue in a standardized exercise test (SET). Total diets provided an average of 1,206.67, 844.61, and 263.13 g of starch/d in HS, MS, and LS, respectively. Horses were lightly exercised for 30 min 3 d/wk. The SET consisted of a 30 min trot in a panel exerciser, followed by 27 min of an incremental high-intensity work on a treadmill. Skeletal muscle biopsies were taken from the biceps femoris at rest, immediately after the SET, and 24 and 48 h post exercise. Samples were submerged in liquid nitrogen and stored at -80ºC until glycogen analysis using a commercial kit. Venous blood samples were taken at rest, immediately post exercise, 10 min after recovery, and 24 h post exercise. Data was analyzed using Proc Mixed (SAS) program. High starch had higher resting muscle glycogen concentration (P = 0.009) than MS (10.25 vs. 8.28 μg/mg wet wt). Low starch had higher glycogen concentration 24 h post (P = 0.04) than HS (9.52 vs. 7.68 μg/mg wet wt). High starch utilized more glycogen than MS or LS. A slight reduction in glycogen post exercise for MS and LS indicated that fat or protein may have been used as substrate for exercise. Results indicated that feeding 1,206.67 g starch/d did not yield an advantage in recovery time over a MS or LS diet. Energy expenditure during the SET yielded similar (P = 0.98) blood lactate concentrations, resulting in the formation of a prediction equation of y = 0.002x^2 – 0.3102x + 6.6874

Development of a gut-on-a-chip model for high throughput disease modeling and drug discovery

A common bottleneck in any drug development process is finding sufficiently accurate models that capture key aspects of disease development and progression. Conventional drug screening models often rely on simple 2D culture systems that fail to recapitulate the complexity of the organ situation. In this study, we show the application of a robust high throughput 3D gut-on-a-chip model for investigating hallmarks of inflammatory bowel disease (IBD). Using the OrganoPlate platform, we subjected enterocyte-like cells to an immune-relevant inflammatory trigger in order to recapitulate key events of IBD and to further investigate the suitability of this model for compound discovery and target validation activities. The induction of inflammatory conditions caused a loss of barrier function of the intestinal epithelium and its activation by increased cytokine production, two events observed in IBD physiopathology. More importantly, anti-inflammatory compound exposure prevented the loss of barrier function and the increased cytokine release. Furthermore, knockdown of key inflammatory regulators RELA and MYD88 through on-chip adenoviral shRNA transduction alleviated IBD phenotype by decreasing cytokine production. In summary, we demonstrate the routine use of a gut-on-a-chip platform for disease-specific aspects modeling. The approach can be used for larger scale disease modeling, target validation and drug discovery purpose

A cluster randomized controlled trial aimed at implementation of local quality improvement collaboratives to improve prescribing and test ordering performance of general practitioners: Study Protocol

<p>Abstract</p> <p>Background</p> <p>The use of guidelines in general practice is not optimal. Although evidence-based methods to improve guideline adherence are available, variation in physician adherence to general practice guidelines remains relatively high. The objective for this study is to transfer a quality improvement strategy based on audit, feedback, educational materials, and peer group discussion moderated by local opinion leaders to the field. The research questions are: is the multifaceted strategy implemented on a large scale as planned?; what is the effect on general practitioners' (GPs) test ordering and prescribing behaviour?; and what are the costs of implementing the strategy?</p> <p>Methods</p> <p>In order to evaluate the effects, costs and feasibility of this new strategy we plan a multi-centre cluster randomized controlled trial (RCT) with a balanced incomplete block design. Local GP groups in the south of the Netherlands already taking part in pharmacotherapeutic audit meeting groups, will be recruited by regional health officers. Approximately 50 groups of GPs will be randomly allocated to two arms. These GPs will be offered two different balanced sets of clinical topics. Each GP within a group will receive comparative feedback on test ordering and prescribing performance. The feedback will be discussed in the group and working agreements will be created after discussion of the guidelines and barriers to change. The data for the feedback will be collected from existing and newly formed databases, both at baseline and after one year.</p> <p>Discussion</p> <p>We are not aware of published studies on successes and failures of attempts to transfer to the stakeholders in the field a multifaceted strategy aimed at GPs' test ordering and prescribing behaviour. This pragmatic study will focus on compatibility with existing infrastructure, while permitting a certain degree of adaptation to local needs and routines.</p> <p>Trial registration</p> <p>Nederlands Trial Register ISRCTN40008171</p