Contractility analysis of human engineered 3D heart tissues by an automatic tracking technique using a standalone application.

The use of Engineered Heart Tissues (EHT) as in vitro model for disease modeling and drug screening has increased, as they provide important insight into the genetic mechanisms, cardiac toxicity or drug responses. Consequently, this has highlighted the need for a standardized, unbiased, robust and automatic way to analyze hallmark physiological features of EHTs. In this study we described and validated a standalone application to analyze physiological features of EHTs in an automatic, robust, and unbiased way, using low computational time. The standalone application "EHT Analysis" contains two analysis modes (automatic and manual) to analyzes the contractile properties and the contraction kinetics of EHTs from high speed bright field videos. As output data, the graphs of displacement, contraction force and contraction kinetics per file will be generated together with the raw data. Additionally, it also generates a summary file containing all the data from the analyzed files, which facilitates and speeds up the post analysis. From our study we highlight the importance of analyzing the axial stress which is the force per surface area (µN/mm2). This allows to have a readout overtime of tissue compaction, axial stress and leave the option to calculate at the end point of an experiment the physiological cross-section area (PSCA). We demonstrated the utility of this tool by analyzing contractile properties and compaction over time of EHTs made out of a double reporter human pluripotent stem cell (hPSC) line (NKX2.5EGFP/+-COUP-TFIImCherry/+) and different ratios of human adult cardiac fibroblasts (HCF). Our standalone application "EHT Analysis" can be applied for different studies where the physiological features of EHTs needs to be analyzed under the effect of a drug compound or in a disease model.

Integrated microfluidic biosensing platform for simultaneous confocal microscopy and electrophysiological measurements on bilayer lipid membranes and ion channels.

Combining high-resolution imaging and electrophysiological recordings is key for various types of experimentation on lipid bilayers and ion channels. Here, we propose an integrated biosensing platform consisting of a microfluidic cartridge and a dedicated chip-holder to conduct such dual measurements on suspended lipid bilayers, in a user-friendly manner. To illustrate the potential of the integrated platform, we characterize lipid bilayers in terms of thickness and fluidity while simultaneously monitoring single ion channel currents. For that purpose, POPC lipid bilayers are supplemented with a fluorescently-tagged phospholipid (NBD-PE, 1% mol) for Fluorescence Recovery After Photobleaching (FRAP) measurements and a model ion channel (gramicidin, 1 nM). These combined measurements reveal that NBD-PE has no effect on the lipid bilayer thickness while gramicidin induces thinning of the membrane. Furthermore, the presence of gramicidin does not alter the lipid bilayer fluidity. Surprisingly, in lipid bilayers supplemented with both probes, a reduction in gramicidin open probability and lifetime is observed compared to lipid bilayers with gramicidin only, suggesting an influence of NBD-PE on the gramicidin ion function. Altogether, our proposed microfluidic biosensing platform in combination with the herein presented multi-parametric measurement scheme paves the way to explore the interdependent relationship between lipid bilayer properties and ion channel function.

High-throughput deterministic single-cell encapsulation and droplet pairing, fusion, and shrinkage in a single microfluidic device.

In this article, we present a microfluidic device capable of successive high-yield single-cell encapsulation in droplets, with additional droplet pairing, fusion, and shrinkage. Deterministic single-cell encapsulation is realized using Dean-coupled inertial ordering of cells in a Yin-Yang-shaped curved microchannel using a double T-junction, with a frequency over 2000 Hz, followed by controlled droplet pairing with a 100% success rate. Subsequently, droplet fusion is realized using electrical actuation resulting in electro-coalescence of two droplets, each containing a single HL60 cell, with 95% efficiency. Finally, volume reduction of the fused droplet up to 75% is achieved by a triple pitchfork structure. This droplet volume reduction is necessary to obtain close cell-cell membrane contact necessary for final cell electrofusion, leading to hybridoma formation, which is the ultimate aim of this research.

Al2O3/silicon nanoISFET with near ideal nernstian response.

Nanoscale ISFET (ion sensitive field-effect transistor) pH sensors are presented that produce the well-known sub-nernstian pH-response for silicon dioxide (SiO(2)) surfaces and near ideal nernstian sensitivity for alumina (Al(2)O(3)) surfaces. Titration experiments of SiO(2) surfaces resulted in a varying pH sensitivity ∼20 mV/pH for pH near 2 and >45 mV/pH for pH > 5. Measured pH responses from titrations of thin (15 nm) atomic layer deposited (ALD) alumina (Al(2)O(3)) surfaces on the nanoISFETs resulted in near ideal nernstian pH sensitivity of 57.8 ± 1.2 mV/pH (pH range: 2-10; T = 22 °C) and temperature sensitivity of 0.19 mV/pH °C (22 °C ≤ T ≤ 40 °C). A comprehensive analytical model of the nanoISFET sensor, which is based on the combined Gouy-Chapman-Stern and Site-Binding (GCS-SB) model, accompanies the experimental results and an extracted ΔpK ≈ 1.5 from the measured responses further supports the near ideal nernstian pH sensitivity.

High-resolution microcontact printing and transfer of massive arrays of microorganisms on planar and compartmentalized nanoporous aluminium oxide.

Handling microorganisms in high throughput and their deployment into miniaturized platforms presents significant challenges. Contact printing can be used to create dense arrays of viable microorganisms. Such "living arrays", potentially with multiple identical replicates, are useful in the selection of improved industrial microorganisms, screening antimicrobials, clinical diagnostics, strain storage, and for research into microbial genetics. A high throughput method to print microorganisms at high density was devised, employing a microscope and a stamp with a massive array of PDMS pins. Viable bacteria (Lactobacillus plantarum, Esherichia coli), yeast (Candida albicans) and fungal spores (Aspergillus fumigatus) were deposited onto porous aluminium oxide (PAO) using arrays of pins with areas from 5 x 5 to 20 x 20 microm. Printing onto PAO with up to 8100 pins of 20 x 20 microm area with 3 replicates was achieved. Printing with up to 200 pins onto PAO culture chips (divided into 40 x 40 microm culture areas) allowed inoculation followed by effective segregation of microcolonies during outgrowth. Additionally, it was possible to print mixtures of C. albicans and spores of A. fumigatus with a degree of selectivity by capture onto a chemically modified PAO surface. High resolution printing of microorganisms within segregated compartments and on functionalized PAO surfaces has significant advantages over what is possible on semi-solid surfaces such as agar.

Viability analysis and apoptosis induction of breast cancer cells in a microfluidic device: effect of cytostatic drugs.

Breast cancer is the leading cause of cancer deaths among non-smoking women worldwide. At the moment the treatment regime is such that patients receive different chemotherapeutic and/or hormonal treatments dependent on the hormone receptor status, the menopausal status and age. However, in vitro sensitivity testing of tumor biopsies could rationalize and improve the choice of chemo- and hormone therapy. Lab-on-a-Chip devices, using microfluidic techniques, make detailed cellular analysis possible using fewer cells, enabling working with a patients' own cells and performing chemo- and hormone sensitivity testing in an ex vivo setting. This article describes the development of two microfluidic devices made in poly(dimethylsiloxane) (PDMS) to validate the cell culture properties and analyze the chemosensitivity of MCF-7 cells (estrogen receptor positive human breast cancer cells) in response to the drug staurosporine (SSP). In both cases, cell viability was assessed using the life-stain Calcein-AM (CAAM) and the death dye propidium iodide (PI). MCF-7 cells could be statically cultured for up to 7 days in the microfluidic chip. A 30 min flow with SSP and a subsequent 24 h static incubation in the incubator induced apoptosis in MCF-7 cells, as shown by a disappearance of the aggregate-like morphology, a decrease in CAAM staining and an increase in PI staining. This work provides valuable leads to develop a microfluidic chip to test the chemosensitivity of tumor cells in response to therapeutics and in this way improve cancer treatment towards personalized medicine.

Bioactive constituents of Cedrelopsis microfoliata.

The stem bark of Cedrelopsis microfoliata has yielded the new compounds microfolian (1), microfolione (2), and microfolicoumarin (6) along with the known compounds agrandol (3), cedrecoumarin A (4), obliquin (5), and sesquichamaenol (7). Compounds 2-4 were found to show agonistic activity on both alpha- and beta-estrogenic receptors, and compounds 1, 3, and 4 were shown to inhibit the chemiluminescence of reactive oxygen metabolites and showed superoxide scavenging activity.