Micro-Engineered Heart Tissues On-Chip with Heterotypic Cell Composition Display Self-Organization and Improved Cardiac Function.

Advanced in vitro models that recapitulate the structural organization and function of the human heart are highly needed for accurate disease modeling, more predictable drug screening, and safety pharmacology. Conventional 3D Engineered Heart Tissues (EHTs) lack heterotypic cell complexity and culture under flow, whereas microfluidic Heart-on-Chip (HoC) models in general lack the 3D configuration and accurate contractile readouts. In this study, an innovative and user-friendly HoC model is developed to overcome these limitations, by culturing human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs), endothelial (ECs)- and smooth muscle cells (SMCs), together with human cardiac fibroblasts (FBs), underflow, leading to self-organized miniaturized micro-EHTs (µEHTs) with a CM-EC interface reminiscent of the physiological capillary lining. µEHTs cultured under flow display enhanced contractile performance and conduction velocity. In addition, the presence of the EC layer altered drug responses in µEHT contraction. This observation suggests a potential barrier-like function of ECs, which may affect the availability of drugs to the CMs. These cardiac models with increased physiological complexity, will pave the way to screen for therapeutic targets and predict drug efficacy.

Automated assessment of human engineered heart tissues using deep learning and template matching for segmentation and tracking.

The high rate of drug withdrawal from the market due to cardiovascular toxicity or lack of efficacy, the economic burden, and extremely long time before a compound reaches the market, have increased the relevance of human in vitro models like human (patient-derived) pluripotent stem cell (hPSC)-derived engineered heart tissues (EHTs) for the evaluation of the efficacy and toxicity of compounds at the early phase in the drug development pipeline. Consequently, the EHT contractile properties are highly relevant parameters for the analysis of cardiotoxicity, disease phenotype, and longitudinal measurements of cardiac function over time. In this study, we developed and validated the software HAARTA (Highly Accurate, Automatic and Robust Tracking Algorithm), which automatically analyzes contractile properties of EHTs by segmenting and tracking brightfield videos, using deep learning and template matching with sub-pixel precision. We demonstrate the robustness, accuracy, and computational efficiency of the software by comparing it to the state-of-the-art method (MUSCLEMOTION), and by testing it with a data set of EHTs from three different hPSC lines. HAARTA will facilitate standardized analysis of contractile properties of EHTs, which will be beneficial for in vitro drug screening and longitudinal measurements of cardiac function.

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.

A fluorescent microscopy-screening test for efficient STR-typing of telogen hair roots.

Nuclear DNA-profiling from human hairs is a well-known technique in forensic investigations, but its success rate is quite low with some hair types. As nuclear DNA (nuDNA) extracted from telogen hair roots is in short supply and is often degraded, a simple and effective method of estimating the number of nuclear DNAs in telogen roots has been developed. DAPI, a fluorescent, non-destructive DNA stain, allows the visualization of "nuclei" (DAPI-positive spots the shape and size of the human follicular cell nuclei) and does not interfere with subsequent PCR analyses. We stained 3242 telogen roots from 27 donors. Surprisingly, of the 2572 club roots without any soft tissue remnants 11% contained visible "nuclei" and 3.3% even contained many. At the same time 57% of the 670 telogen roots with soft tissue remnants did not show any fluorescent "nuclei". We analysed the STR-profile of some of the roots selected by the DAPI screening, i.e. 132 telogen roots without soft tissue remnants, with a success rate of 79%. Our proposed screening method allows the DNA laboratory to analyse nuclear DNA only in the most promising hair roots.

Toxicological data and growth characteristics of single post-feeding larvae and puparia of Calliphora vicina (Diptera: Calliphoridae) obtained from a controlled nordiazepam study.

Larvae of the Calliphora vicina (Diptera: Calliphoridae) were reared on artificial food spiked with different concentrations of nordiazepam. The dynamics of the accumulation and conversion of nordiazepam to its metabolite oxazepam in post-feeding larvae and empty puparia were studied. Analysis was performed using a previously developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. This method enabled the detection and quantitation of nordiazepam and oxazepam in single larvae and puparia. Both drugs could be detected in post-feeding larvae and empty puparia. In addition, the influence of nordiazepam on the development and growth of post-feeding larvae was studied. However, no major differences were observed for these parameters between the larvae fed on food containing nordiazepam and the control group. To our knowledge, this is the first report describing the presence of nordiazepam and its metabolite, oxazepam, in single Calliphora vicina larvae and puparia.