Well-plate Format Research Articles

High throughput screening for SARS-CoV-2 helicase inhibitors

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for nearly 7 million deaths worldwide since its outbreak in late 2019. Even with the rapid development and production of vaccines and intensive research, there is still a huge need for specific anti-viral drugs that address the rapidly arising new variants. To address this concern, the National Institute of Allergy and Infectious Diseases (NIAID) established nine Antiviral Drug Discovery (AViDD) Centers, tasked with exploring approaches to target pathogens with pandemic potential, including SARS-CoV-2. In this study, we sought inhibitors of SARS-CoV2 non-structural protein 13 (nsP13) as potential antivirals, first developing a HTS-compatible assay to measure SARS-CoV2 nsP13 helicase activity. Here we present our effort in implementing the assay in a 1,536 well-plate format and in identifying nsP13 inhibitor hit compounds from a ∼650,000 compound library. The primary screen was robust (average Z’ = 0.86 ± 0.05) and resulted in 7,009 primary hits. 1,763 of these compounds upon repeated retests were further confirmed, showing consistent inhibition. Following in-silico analysis, an additional orthogonal assay and titration assays, we identified 674 compounds with IC50 <10 μM. We confirmed activity of independent compound batches from de novo powders while also incorporating multiple counterscreen assays. Our study highlights the potential of this assay for use on HTS platforms to discover novel compounds inhibiting SARS-CoV2 nsP13, which merit further development as an effective SARS-CoV2 antiviral.

Simultaneous screening for selective SARS-CoV-2, Lassa, and Machupo virus entry inhibitors

Emerging highly pathogenic viruses can pose profound impacts on global health, the economy, and society. To meet that challenge, the National Institute of Allergy and Infectious Diseases (NIAID) established nine Antiviral Drug Discovery (AViDD) centers for early-stage identification and validation of novel antiviral drug candidates against viruses with pandemic potential. As part of this initiative, we established paired entry assays that simultaneously screen for inhibitors specifically targeting SARS-CoV-2 (SARS2), Lassa virus (LASV) and Machupo virus (MACV) entry. To do so we employed a dual pseudotyped virus (PV) infection system allowing us to screen ∼650,000 compounds efficiently and cost-effectively. Adaptation of these paired assays into 1536 well-plate format for ultra-high throughput screening (uHTS) resulted in the largest screening ever conducted in our facility, with over 2.4 million wells completed. The paired infection system allowed us to detect two PV infections simultaneously: LASV + MACV, MACV + SARS2, and SARS2 + LASV. Each PV contains a different luciferase reporter gene which enabled us to measure the infection of each PV exclusively, albeit in the same well. Each PV was screened at least twice utilizing different reporters, which allowed us to select the inhibitors specific to a particular PV and to exclude those that hit off targets, including cellular components or the reporter proteins. All assays were robust with an average Z’ value ranging from 0.5 to 0.8. The primary screening of ∼650,000 compounds resulted in 1812, 1506, and 2586 unique hits for LASV, MACV, and SARS2, respectively. The confirmation screening narrowed this list further to 60, 40, and 90 compounds that are unique to LASV, MACV, and SARS2, respectively. Of these compounds, 8, 35, and 50 compounds showed IC50 value < 10 μM, some of which have much greater potency and excellent antiviral activity profiles specific to LASV, MACV, and SARS2, and none are cytotoxic. These selected compounds are currently being studied for their mechanism of action and to improve their specificity and potency through chemical modification.

Portable fiber-based double nanohole optical tweezer for trapping small proteins

We demonstrate the trapping and analysis of individual proteins using a portable optical fiber tweezer setup with a double-nanohole in a gold film coating the fiber’s end and aligned with the fiber core. The instrument was used to trap and analyze cytochrome c, carbonic anhydrase, bovine serum albumin, and PR65 (wild-type and various point mutants). This approach was compared with a free-space optical tweezer setup that requires alignment of the laser focus to the aperture, whereas the fiber-based approach is both portable and alignment-free, which holds promise for applications in antibody discovery, small molecule drug discovery, protein interaction analysis and other applications using the standard well-plate format.

Tunable In Situ Synthesis of Ultrathin Extracellular Matrix-Derived Membranes in Organ-on-a-Chip Devices.

Thin cell culture membranes in organ-on-a-chip (OOC) devices are used to model a wide range of thin tissues. While early and most current platforms use microporous or fibrous elastomeric or thermoplastic membranes, there is an emerging class of devices using extra-cellular matrix (ECM) protein-based membranes to improve their biological relevance. These ECM-based membranes present physiologically relevant properties, but they are difficult to integrate into OOC devices due to their relative fragility. Additionally, the specialized fabrication methods developed to date make comparison between methods difficult. This work presents the development and characterization of a method to produce ultrathin matrix-derived membranes (UMM) in OOC devices that requires only a preassembled thermoplastic device and a micropipette, decoupling the device and UMM fabrication processes. Control over the thickness and permeability of the UMM is demonstrated, along with integration of the UMM in a device enabling high-resolution on-chip microscopy. The reliability of the UMM fabrication method is leveraged to develop a medium-throughput well-plate format device with 32 independent UMM-integrated samples. Finally, proof-of-concept cell culture experiments are demonstrated. Due to its simplicity and controllability, the presented method has the potential to overcome technical barriers preventing wider adoption of physiologically relevant ECM-based membranes in OOC devices.

Abstract 7288: Evaluation of silica spin-column and bead formats for rapid promoter DNA methylation analysis by qMSP in clinical and point-of-care settings

Abstract Late-stage cancers often lack effective management and treatment options, emphasizing the importance of early diagnosis for better prognosis and lower mortality rates. Molecular markers, such as DNA methylation tests, have shown promise in predicting and detecting cancer at its earliest stages. The COVID-19 post-pandemic scenario is providing a one in one-hundred-year opportunity of utilizing the excess installed PCR capacity world-wide to develop novel Quantitative Methylation Specific PCR (qMSP) screening strategies for cervical cancer screening in low-resource settings. This study aimed to compare the use of commercial bisulfite conversion kits in cervical epithelium liquid cytology samples (liquid cytology) from cancer screening clinics in the United States. Commercially available rapid bisulfite conversion protocols using silica spin column and magnetic beads were compared with conventional DNA extraction followed by bisulfite conversion protocols to profile DNA methylation by qMSP in a panel of methylated genes we previously developed to distinguish normal and cervical cancer patient samples. The results revealed that small sample volumes are suitable for methylation studies. Comparisons among four spin-column kits showed no significant differences in β-actin Cycle Threshold (Cts) values between Abcam, Epigentek, QIAGEN and Zymo D5021 kits. Zymo The best performing spin-column kit was compared with manual and automated versions of magnetic bead kits using a 96-well plate format for higher throughput. Methylation profiling of three genes (β-actin ZNF516, and FKBP6) with manual and automated magnetic bead kits revealed that automation increases throughput without a reduction in amplification efficiency in open PCR systems. Cost analysis showed that the direct kits 96-well plate format were more affordable compared to the Gold Standard manual protocol that uses spin-columns. An optimized version of Zymo D5046 magnetic bead kit provides the fastest turnaround time with similar amplification efficiency, when compared to spin-column or other magnetic bead kits. The test cost per sample is $5.41 dollars to process 83 samples in a 96 well-plate format in less than 2 hours. Another significant finding was showing the feasibility of using as low as 12µL of liquid cytology samples for DNA methylation testing, proving that bodily fluids are a liquid biopsy alternative to plasma. Overall, this comprehensive comparison is useful in determining optimal options for efficient and cost-effective cancer DNA methylation diagnostic tests, particularly in low-resource settings. Citation Format: Fernando Zamuner, Ashley Ramos-Lopez, Amanda Garcia-Negron, Ana Purcell-Wiltz, Andrea Cortes-Ortiz, Aniris Roman, Keerthana Gosala, Eli Winkler, David Sidransky, Mariana Brait, Rafael E. Guerrero-Preston. Evaluation of silica spin-column and bead formats for rapid promoter DNA methylation analysis by qMSP in clinical and point-of-care settings [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7288.

Microcavity well-plate for automated parallel bioelectronic analysis of 3D cell cultures

Three-dimensional (3D) in vitro cell culture models serve as valuable tools for accurately replicating cellular microenvironments found in vivo. While cell culture technologies are rapidly advancing, the availability of non-invasive, real-time, and label-free analysis methods for 3D cultures remains limited. To meet the demand for higher-throughput drug screening, there is a demanding need for analytical methods that can operate in parallel. Microelectrode systems in combination with microcavity arrays (MCAs), offer the capability of spatially resolved electrochemical impedance analysis and field potential monitoring of 3D cultures. However, the fabrication and handling of small-scale MCAs have been labour-intensive, limiting their broader application. To overcome this challenge, we have established a process for creating MCAs in a standard 96-well plate format using high-precision selective laser etching. In addition, to automate and ensure the accurate placement of 3D cultures on the MCA, we have designed and characterized a plug-in tool using SLA-3D-printing. To characterize our new 96-well plate MCA-based platform, we conducted parallel analyses of human melanoma 3D cultures and monitored the effect of cisplatin in real-time by impedance spectroscopy. In the following we demonstrate the capabilities of the MCA approach by analysing contraction rates of human pluripotent stem cell-derived cardiomyocyte aggregates in response to cardioactive compounds. In summary, our MCA system significantly expands the possibilities for label-free analysis of 3D cell and tissue cultures, offering an order of magnitude higher parallelization capacity than previous devices. This advancement greatly enhances its applicability in real-world settings, such as drug development or clinical diagnostics.

FRESH™ 3D bioprinted cardiac tissue, a bioengineered platform for in vitro pharmacology.

There is critical need for a predictive model of human cardiac physiology in drug development to assess compound effects on human tissues. In vitro two-dimensional monolayer cultures of cardiomyocytes provide biochemical and cellular readouts, and in vivo animal models provide information on systemic cardiovascular response. However, there remains a significant gap in these models due to their incomplete recapitulation of adult human cardiovascular physiology. Recent efforts in developing in vitro models from engineered heart tissues have demonstrated potential for bridging this gap using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in three-dimensional tissue structure. Here, we advance this paradigm by implementing FRESH™ 3D bioprinting to build human cardiac tissues in a medium throughput, well-plate format with controlled tissue architecture, tailored cellular composition, and native-like physiological function, specifically in its drug response. We combined hiPSC-CMs, endothelial cells, and fibroblasts in a cellular bioink and FRESH™ 3D bioprinted this mixture in the format of a thin tissue strip stabilized on a tissue fixture. We show that cardiac tissues could be fabricated directly in a 24-well plate format were composed of dense and highly aligned hiPSC-CMs at >600 million cells/mL and, within 14 days, demonstrated reproducible calcium transients and a fast conduction velocity of ∼16 cm/s. Interrogation of these cardiac tissues with the β-adrenergic receptor agonist isoproterenol showed responses consistent with positive chronotropy and inotropy. Treatment with calcium channel blocker verapamil demonstrated responses expected of hiPSC-CM derived cardiac tissues. These results confirm that FRESH™ 3D bioprinted cardiac tissues represent an in vitro platform that provides data on human physiological response.

Abstract 4560: Assay-ready tissue-engineered cancer microspheres for high-throughput screening

Abstract For drug discovery, new in vitro cancer models are needed to obtain more translatable study outcomes in a low-cost and high-throughput manner. For this purpose, 3D cancer spheroids have been established as more effective than 2D models. Current commercial techniques, however, rely heavily on self-aggregation of dissociated cells and cannot replicate key features of the native tumor microenvironment, particularly due to a lack of control over extracellular matrix components and heterogeneity in size and aggregate-forming tendencies. Also, current spheroidal techniques are typically limited to one spheroid per well, therefore providing a narrow range of cell numbers per well, disadvantageous for assay development in drug screening. Here, we overcome these challenges by coupling tissue engineering toolsets with microfluidic technologies to create engineered cancer microspheres and sorting desired numbers of microspheres into assay-ready well-plate format. To form the engineered cancer microspheres, MCF7 (non-metastatic) and MDA-MB-231 (metastatic) breast cancer cells were encapsulated within poly(ethylene glycol)-fibrinogen hydrogels using our previously developed microfluidic platform. Highly uniform cancer microspheres (intra and inter-batch coefficient of variation ≤ 5%) with high cell densities (over 20 × 106 cells/ml) were produced rapidly, which is critical for use in drug testing. The microspheres supported the 3D culture of both breast cancer cell lines over at least 14 days in culture. Encapsulated cells displayed cell type-specific differences in morphology, proliferation, metabolic activity, ultrastructure, and overall microsphere size distribution and bulk stiffness. To prepare assay-ready pre-plated microspheres, a COPAS FP flow cytometer was used for its ability to analyze and sort large sample particles such as tumor spheroids and hydrogel cancer microspheres generated in this study. When using a 96-well plate, the sorting rate varied from 2.5 - 6 microspheres per second, depending on the sample concentration. When sorting a desired number of microspheres per well, the accuracy was greater than 95% as verified visually by microscopy. Viability of sorted microspheres was verified 24 hours post-sort. Shipping conditions were established that maintained cell viability for remote use in drug testing. Methods for compound addition by pinning and imaging were tested and optimized. Using these approaches, the microsphere system was shown to be compatible with an automated liquid handling system for administration of drug compounds; MDA-MB-231 microspheres were distributed in 384 well plates and treated with chemotherapeutic drugs. Expected responses were quantitated using CellTiter-Glo® 3D and detected using automated imaging. Overall, our results demonstrate initial applicability for the tissue-engineered cancer microspheres for drug screening. Citation Format: Yuan Tian, Yongwoon Kim, Wen J. Seeto, Shantanu Pradhan, Dmitriy Minond, Rock Pulak, Elizabeth A. Lipke. Assay-ready tissue-engineered cancer microspheres for high-throughput screening. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4560.

Xanthate-supported photo-iniferter (XPI)-RAFT polymerization: facile and rapid access to complex macromolecules.

Xanthate-supported photo-iniferter (XPI)-reversible addition-fragmentation chain-transfer (RAFT) polymerization is introduced as a fast and versatile photo-polymerization strategy. Small amounts of xanthate are added to conventional RAFT polymerizations to act as a photo-iniferter under light irradiation. Radical exchange is facilitated by the main CTA ensuring control over the molecular weight distribution, while xanthate enables an efficient photo-(re)activation. The photo-active moiety is thus introduced into the polymer as an end group, which makes chain extension of the produced polymers possible directly by irradiation. This is in sharp contrast to conventional photo-initiators, or photo electron transfer (PET)-RAFT polymerizations, where radical generation depends on the added small molecules. In contrast to regular photo-iniferter-RAFT polymerization, photo-activation is decoupled from polymerization control, rendering XPI-RAFT an elegant tool for the fabrication of defined and complex macromolecules. The method is oxygen tolerant and robust and was used to perform screenings in a well-plate format, and it was even possible to produce multiblock copolymers in a coffee mug under open-to-air conditions. XPI-RAFT does not rely on highly specialized equipment and qualifies as a universal tool for the straightforward synthesis of complex macromolecules. The method is user-friendly and broadens the scope of what can be achieved with photo-polymerization techniques.

High-Efficiency Multi-Drug Functional Profiling in a Microfluidic Device Towards Personalized Predictions in Pediatric Leukemia

Treatment of pediatric acute lymphoblastic leukemia (ALL) has seen dramatic improvements over the last several decades, leading to overall survival rates of over 85%. However, for patients with relapsed/refractory disease, achieving complete remission remains extremely challenging. Improving treatment outcomes for these patients will require increasingly personalized treatment strategies that make use of functional screens in addition to molecular profiling technologies for treatment selection. Given the past success of combination chemotherapy in cancer treatment and recent promise for newly developed targeted agents, functional assays that are able to evaluate patient response to combinations of therapies will likely be most effective in identifying clinically actionable treatment protocols. Moreover, assays that can achieve this with minimal patient sample and reduced experimental workload will have the greatest potential for clinical translation and integration into clinical workflows. To that end, this work aims to develop a microfluidic assay to directly test candidate combinations of therapeutics in patient derived ALL cells. We aim for this to serve as a more effective way to screen for drug combination response and higher order interactions relative to standard wellplate formats. Once validated, this assay can be used to identify biomarkers of response to standard and experimental combination regimens in pediatric ALL, and ultimately, could be used to prospectively guide therapy in newly diagnosed patients. The developed device achieves this by enabling simultaneous exposure to 3 superimposed small molecule drug gradients across a 3D culture of leukemia cells such that each location in the device comprises a unique combination of all 3 drugs within a simplex (Fig 1A). Single cell drug concentration exposure is determined from experimentally validated steady state computational fluid dynamics (CFD) model predictions, and single cell viability is analyzed via Calcein (live), PI (dead) and Hoechst (nuclear) staining following 48 hours of treatment. Using this system, we then develop a novel experimental and computational framework for evaluating drug combination efficacy and synergy across the full range of combination ratios captured in our device. Specifically, we evaluate response across multiple devices at optimally spaced dose levels, increasing coverage of the 3-drug combination space and allowing for custom implementation of established synergy models. This enables synergy assessment in a continuous gradient of drug combination ratios, as opposed to the discrete combination ratios commonly evaluated in standard assays, and may further improve detection of drug synergy and identification of the most potent and efficacious combination ratios. To demonstrate this, we analyze response to a combination of daunorubicin, vincristine, and nelarabine in Jurkats, a T-ALL cell line, across 7 different devices at 7 different dose levels (Fig 1B). We quantify viability as a function of concentration across each device and each dose level, and generate dose response curves at distinct zones within the device. Specifically, we generate dose response curves in zones dominated by daunorubicin, vincristine, and nelarabine alone and find half maximal effective concentration values (EC50 values) similar to those reported in literature. We further evaluate response at varying ratios of each 2-drug combination, and generate dose response curves as shown in Fig 1B. Analyzing the 2-drug combination dose responses relative to single-drug dose responses allows for quantification of 2-drug combination synergy based on Loewe and DiaMOND synergy models, and future work will extend our developed method to analyze emergent 3-drug combination synergy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High-Throughput Dispensing of Viscous Solutions for Biomedical Applications

Cells cultured in three-dimensional scaffolds express a phenotype closer to in vivo cells than cells cultured in two-dimensional containers. Natural polymers are suitable materials to make three-dimensional scaffolds to develop disease models for high-throughput drug screening owing to their excellent biocompatibility. However, natural polymer solutions have a range of viscosities, and none of the currently available liquid dispensers are capable of dispensing highly viscous polymer solutions. Here, we report the development of an automated scaffold dispensing system for rapid, reliable, and homogeneous creation of scaffolds in well-plate formats. We employ computer-controlled solenoid valves to regulate air pressure impinging upon a syringe barrel filled with scaffold solution to be dispensed. Automated dispensing of scaffold solution is achieved via a programmable software interface that coordinates solution extrusion and the movement of a dispensing head. We show that our pneumatically actuated dispensing system can evenly distribute high-viscosity, chitosan-based polymer solutions into 96- and 384-well plates to yield highly uniform three-dimensional scaffolds after lyophilization. We provide a proof-of-concept demonstration of high-throughput drug screening by culturing glioblastoma cells in scaffolds and exposing them to temozolomide. This work introduces a device that can hasten the creation of three-dimensional cell scaffolds and their application to high-throughput testing.

L-lactate as an indicator for cellular metabolic status: An easy and cost-effective colorimetric L-lactate assay.

BackgroundIn recent times, the study of metabolic pathways has become inevitable and predominant for a variety of research fields as cancer biology and immunology. L-lactate as a product of anaerobic glycolysis has shown to be an important indicator of the cellular metabolic status and can be associated with diverse cellular effects. For this reason, L-lactate assay kits are of high demand when metabolic effects need to be considered. Nevertheless, commercially available kits are not affordable if multiple samples must be evaluated.Principal findingIn this work, we develop an easy and cost-effective colorimetric assay for quantification of L-lactate suitable for cells with low or high L-lactate production based on LDH activity and suitable for 96 well-plate format. Using different metabolic regulators, we demonstrate the capacity of the assay to detect and quantify L-lactate from the supernatant of HeLa cancer cell line. Furthermore, we validate the assay against a commercially available kit by demonstrating no significant difference between both assays. Finally, we show that the assay is capable of quantifying L-lactate in primary cells such as hPBMCs that were stimulated with toll-like receptor ligands and treated with different metabolic regulators.ConclusionWe herein present an easy custom assay that is suitable for cells with low and high L-lactate production at very low cost compared to commercially available kits. These advantages of the custom assay can simplify the research in the field of metabolism and related fields.

Development and Validation of an Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry Method for Quantitation of Total 3,3’,5-triiodo-L-Thyronine and 3,3’,5,5’-tetraiodo-L-Thyronine in Rodent Serum

Many environmental chemicals are known to disrupt thyroid function. Measurement of thyroid hormones in animal studies provides useful information to understand the effects of environmental chemicals on thyroid hormone metabolism. We report an efficient method, utilizing a protein precipitation followed by ultraperformance liquid chromatography-tandem mass spectrometry analysis, to quantitate total 3,3’,5-triiodo-L-thyronine (triiodothyronine, T3) and total 3,3’,5,5’-tetraiodo-L-thyronine (thyroxine, T4) in rodent serum. The use of synthetic serum for calibration standards eliminated the interferences from endogenous total T3 and T4 and allowed the experimental lower limits of quantitation (LOQ) to be set at the required concentration (T3, 20 ng/dL; T4, 0.5 µg/dL) to allow quantitation of endogenous concentrations. The method was linear (r > 0.99; range 20.0-600 ng/dL T3, 0.500-15 µg/dL T4) with good assay recoveries (90.4-107%) for both analytes. Intra- and inter-day accuracy, estimated as percent relative error, were ≤ ±7.6% and intra- and inter-day precision, estimated as the relative standard deviation, were ≤ 5.3% for both analytes. The method may easily be adapted to a well-plate format thereby further improving the efficiency. Total T3 and T4 concentrations were stable in male and female rat and mouse serum when stored in the freezer (∼−70 °C) for up to 62 d with determined values within 92.8-111% of day 0 for both analytes. The method can be extended to quantitate total T3 and T4 concentrations in humans or other species with minimal optimization.

Development and validation of an LC–MS/MS generic assay platform for small molecule drug bioanalysis

AimWe developed a generic high-performance liquid chromatography mass spectrometry approach for quantitation of small molecule compounds without availability of isotopically labelled standard. MethodsThe assay utilized 50 μL of plasma and offers 8 potential internal standards (IS): acetaminophen, veliparib, busulfan, neratinib, erlotinib, abiraterone, bicalutamide, and paclitaxel. Preparation consisted of acetonitrile protein precipitation and aqueous dilution in a 96 well-plate format. Chromatographic separation was achieved with a Kinetex C18 reverse phase (2.6 μm, 2 mm x 50 mm) column and a gradient of 0.1 % formic acid in acetonitrile and water over an 8 min run time. Mass spectrometric detection was performed on an AB SCIEX4000QTRAP with electrospray, positive-mode ionization. Performance of the generic approach was evaluated with seven drugs (LMP744, olaparib, cabozantinib, triapine, ixabepilone, berzosertib, eribulin) for which validated assays were available. ResultsThe 8 IS covered a range of polarity, size, and ionization; eluted over the range of chromatographic retention times; were quantitatively extracted; and suffered limited matrix effects. The generic approach proved to be linear for test drugs evaluated over at least 3 orders of magnitude starting at 1−10 ng/mL, with extension of assay ranges with analyte isotopologue MRM channels. At a bias of less than 16 % and precision within 15 %, the assay performance was acceptable. ConclusionThe generic approach has become a useful tool to further define the pharmacology of drugs studied in our laboratory and may be utilized as described, or as starting point to develop drug-specific assays with more extensive performance characterization.

Measurement of Free Testosterone in Serum Using Equilibrium DialysisCoupled With ID-UHPLC-MS/MS: Comparison Between Equilibrium Devices

Free testosterone (FT) has been used as a biomarker in clinical patient care and public health research to assess and manage patients with androgenic abnormalities. The latest Endocrine Society clinical practice guideline for testosterone therapy in men with hypogonadism recommends measuring FT for those with borderline and low total testosterone concentrations, or those who have conditions that change SHBG concentrations, such as some metabolic or hormonal diseases, certain medication use, or SHBG genetic polymorphisms. Measuring FT is technically challenging and shows high variability. The CDC clinical standardization program is developing a high throughput method using the gold-standard equilibrium dialysis (ED) procedure with isotope dilution ultra-high-performance liquid chromatography tandem mass spectrometry (ID-UHPLC-MS/MS).A serum sample was dialyzed against a protein-free HEPES buffer (pH 7.4) at 37 °C until equilibrium. After isolating endogenous FT from protein-bound testosterone by ED, isotope-labeled internal standard (13C3-testosterone) was added to the dialysate for quantification. Certified pure primary reference material (National Measurement Institute M914) was used to prepare calibrators, enabling traceable quantitation and ensuring measurement trueness. FT was further isolated from the dialysate matrix using supported liquid extraction and a chromatographic separation from interfering compounds and quantitation by tandem MS. The dialysis step requires maintaining the endogenous free hormone equilibrium so that results in dialysate reflect FT concentrations in the blood without influence from dilution, temperature, or pH. The dialyzer system has a 1:1 sample-to-buffer volume and has been used in reference measurement procedures for free hormone measurements, serving as the standard for method performance comparison. Four commercially available devices designed for high throughput in a multiple well-plate format, requiring respective sample-to-buffer ratios, were evaluated for their recovery, speed, ease of automation by a liquid handling system, repeatability, and robustness. Preliminary results showed that a device with 1:1 sample-to-buffer volume had the most comparable results to those obtained from the standard dialyzer, with the mean bias less than 15%. The device with the highest sample-to-buffer ratio showed bias as high as 50%. These data suggest that controlling sample-to-buffer ratio is a critical step in ED FT method.

A Microfluidic Multisize Spheroid Array for Multiparametric Screening of Anticancer Drugs and Blood-Brain Barrier Transport Properties.

Physiological‐relevant in vitro tissue models with their promise of better predictability have the potential to improve drug screening outcomes in preclinical studies. Despite the advances of spheroid models in pharmaceutical screening applications, variations in spheroid size and consequential altered cell responses often lead to nonreproducible and unpredictable results. Here, a microfluidic multisize spheroid array is established and characterized using liver, lung, colon, and skin cells as well as a triple‐culture model of the blood‐brain barrier (BBB) to assess the effects of spheroid size on (a) anticancer drug toxicity and (b) compound penetration across an advanced BBB model. The reproducible on‐chip generation of 360 spheroids of five dimensions on a well‐plate format using an integrated microlens technology is demonstrated. While spheroid size‐related IC50 values vary up to 160% using the anticancer drugs cisplatin (CIS) or doxorubicin (DOX), reduced CIS:DOX drug dose combinations eliminate all lung microtumors independent of their sizes. A further application includes optimizing cell seeding ratios and size‐dependent compound uptake studies in a perfused BBB model. Generally, smaller BBB‐spheroids reveal an 80% higher compound penetration than larger spheroids while verifying the BBB opening effect of mannitol and a spheroid size‐related modulation on paracellular transport properties.

Predicting exposure concentrations of chemicals with a wide range of volatility and hydrophobicity in different multi-well plate set-ups

Quantification of chemical toxicity in small-scale bioassays is challenging owing to small volumes used and extensive analytical resource needs. Yet, relying on nominal concentrations for effect determination maybe erroneous because loss processes can significantly reduce the actual exposure. Mechanistic models for predicting exposure concentrations based on distribution coefficients exist but require further validation with experimental data. Here we developed a complementary empirical model framework to predict chemical medium concentrations using different well-plate formats (24/48-well), plate covers (plastic lid, or additionally aluminum foil or adhesive foil), exposure volumes, and biological entities (fish, algal cells), focusing on the chemicals’ volatility and hydrophobicity as determinants. The type of plate cover and medium volume were identified as important drivers of volatile chemical loss, which could accurately be predicted by the framework. The model focusing on adhesive foil as cover was exemplary cross-validated and extrapolated to other set-ups, specifically 6-well plates with fish cells and 24-well plates with zebrafish embryos. Two case study model applications further demonstrated the utility of the empirical model framework for toxicity predictions. Thus, our approach can significantly improve the applicability of small-scale systems by providing accurate chemical concentrations in exposure media without resource- and time-intensive analytical measurements.

An on-chip wound healing assay fabricated by xurography for evaluation of dermal fibroblast cell migration and wound closure

Dermal fibroblast cell migration is a key process in a physiological wound healing. Therefore, the analysis of cell migration is crucial for wound healing research. In this study, lab-on-a-chip technology was used to investigate the effects of basic fibroblast growth factor (bFGF), mitomycin C (MMC), MEK1/2 inhibitor (U0126) and fetal calf serum (FCS) on human dermal fibroblast cell migration. The microdevice was fabricated consisting of microchannels, pneumatic lines and pneumatically-activated actuators by xurographic rapid prototyping. In contrast to current approaches in in vitro wound healing such as scratch assays and silicone inserts in wellplate format, which show high variability and poor reproducibility, the current system aims to automate the wounding procedure at high precision and reproducibility using lab-on-a-chip. Traumatic wounding was simulated on-chip on fibroblast cell monolayers by applying air pressure on the flexible circular membrane actuator. Wound closure was monitored using light microscopy and cell migration was evaluated using image analysis. The pneumatically controlled system generates highly reproducible wound sizes compared to the conventional wound healing assay. As proof-of-principle study wound healing was investigated in the presence of several stimulatory and inhibitory substances and culture including bFGF, MMC, U0126 MEK1/2 inhibitor as well as serum starvation to demonstrate the broad applicability of the proposed miniaturized culture microsystem.

Programmable µChopper Device with On-Chip Droplet Mergers for Continuous Assay Calibration.

While droplet-based microfluidics is a powerful technique with transformative applications, most devices are passively operated and thus have limited real-time control over droplet contents. In this report, an automated droplet-based microfluidic device with pneumatic pumps and salt water electrodes was developed to generate and coalesce up to six aqueous-in-oil droplets (2.77 nL each). Custom control software combined six droplets drawn from any of four inlet reservoirs. Using our μChopper method for lock-in fluorescence detection, we first accomplished continuous linear calibration and quantified an unknown sample. Analyte-independent signal drifts and even an abrupt decrease in excitation light intensity were corrected in real-time. The system was then validated with homogeneous insulin immunoassays that showed a nonlinear response. On-chip droplet merging with antibody-oligonucleotide (Ab-oligo) probes, insulin standards, and buffer permitted the real-time calibration and correction of large signal drifts. Full calibrations (LODconc = 2 ng mL−1 = 300 pM; LODamt = 5 amol) required <1 min with merely 13.85 nL of Ab-oligo reagents, giving cost-savings 160-fold over the standard well-plate format while also automating the workflow. This proof-of-concept device—effectively a microfluidic digital-to-analog converter—is readily scalable to more droplets, and it is well-suited for the real-time automation of bioassays that call for expensive reagents.

Bactericidal Activity of Lipid-Shelled Nitric Oxide-Loaded Microbubbles.

The global pandemic of antibiotic resistance is an ever-burgeoning public health challenge, motivating the development of adjunct bactericidal therapies. Nitric oxide (NO) is a potent bioactive gas that induces a variety of therapeutic effects, including bactericidal and biofilm dispersion properties. The short half-life, high reactivity, and rapid diffusivity of NO make therapeutic delivery challenging. The goal of this work was to characterize NO-loaded microbubbles (MB) stabilized with a lipid shell and to assess the feasibility of antibacterial therapy in vitro. MB were loaded with either NO alone (NO-MB) or with NO and octafluoropropane (NO-OFP-MB) (9:1 v/v and 1:1 v/v). The size distribution and acoustic attenuation coefficient of NO-MB and NO-OFP-MB were measured. Ultrasound-triggered release of the encapsulated gas payload was demonstrated with 3-MHz pulsed Doppler ultrasound. An amperometric microelectrode sensor was used to measure NO concentration released from the MB and compared to an NO-OFP-saturated solution. The effect of NO delivery on the viability of planktonic (free living) Staphylococcus aureus (SA) USA 300, a methicillin-resistant strain, was evaluated in a 96 well-plate format. The co-encapsulation of NO with OFP increased the total volume and attenuation coefficient of MB. The NO-OFP-MB were destroyed with a clinical ultrasound scanner with an output of 2.48 MPa peak negative pressure (in situ MI of 1.34) but maintained their echogenicity when exposed to 0.02 MPa peak negative pressure (in situ MI of 0.01. The NO dose in NO-MB and NO-OFP-MB was more than 2-fold higher than the NO-OFP-saturated solution. Delivery of NO-OFP-MB increased bactericidal efficacy compared to the NO-OFP-saturated solution or air and OFP-loaded MB. These results suggest that encapsulation of NO with OFP in lipid-shelled MB enhances payload delivery. Furthermore, these studies demonstrate the feasibility and limitations of NO-OFP-MB for antibacterial applications.