Effect of Surface Patterning and Presence of Collagen I on the Phenotypic Changes of Embryonic Stem Cell Derived Cardiomyocytes

Embryonic stem cell derived cardiomyocytes have been widely investigated for stem cell therapy or in vitro model systems. This study examines how two specific biophysical stimuli, collagen I and cell alignment, affect the phenotypes of embryonic stem cell derived cardiomyocytes in vitro. Three phenotypic indicators are assessed: sarcomere organization, cell elongation, and percentage of binucleation. Murine embryonic stem cells were differentiated in a hanging drop assay and cardiomyocytes expressing GFP-α-actinin were isolated by fluorescent sorting. First, the effect of collagen I was investigated. Addition of soluble collagen I markedly reduced binucleation as a result of an increase in cytokinesis. Laden with a collagen gel layer, myocyte mobility and cell shape change were impeded. Second, the effect of cell alignment by microcontact printing and nanopattern topography was investigated. Both patterning techniques induced cell alignment and elongation. Microcontact printing of 20 μm line pattern accelerated binucleation and nanotopography with 700 nm ridges and 3.5 μm grooves negatively regulated binucleation. This study highlights the importance of biophysical cues in the morphological changes of differentiated cardiomyocytes and may have important implications on how these cells incorporate into the native myocardium.Singapore-MIT Alliance for Research and TechnologyNational Science Foundation (U.S.) ((Science and Technology Center (EBICS): Emergent Behaviors of Integrated Cellular Systems, Grant CBET-0939511)Charles Stark Draper Laboratory (Internal Research and Development Program

Hot embossing for fabrication of a microfluidic 3D cell culture

Clinically relevant studies of cell function in vitro require a physiologically-representative microenvironment possessing aspects such as a 3D extracellular matrix (ECM) and controlled biochemical and biophysical parameters. A polydimethylsiloxane (PDMS) microfluidic system with a 3D collagen gel has previously served for analysis of factors inducing different responses of cells in a 3D microenvironment under controlled biochemical and biophysical parameters. In the present study, applying the known commercially-viable manufacturing methods to a cyclic olefin copolymer (COC) material resulted in a microfluidic device with enhanced 3D gel capabilities, controlled surface properties, and improved potential to serve high-volume applications. Hot embossing and roller lamination molded and sealed the microfluidic device. A combination of oxygen plasma and thermal treatments enhanced the sealing, ensured proper placement of the 3D gel, and created controlled and stable surface properties within the device. Culture of cells in the new device indicated no adverse effects of the COC material or processing as compared to previous PDMS devices. The results demonstrate a methodology to transition microfludic devices for 3D cell culture from scientific research to high-volume applications with broad clinical impact.National Cancer Institute (U.S.) (award R21CA140096)Charles Stark Draper Laboratory (IR&D Grant

Sleep Health, Individual Characteristics, Lifestyle Factors, and Marathon Completion Time in Marathon Runners: A Retrospective Investigation of the 2016 London Marathon

Despite sleep health being critically important for athlete performance and well-being, sleep health in marathoners is understudied. This foundational study explored relations between sleep health, individual characteristics, lifestyle factors, and marathon completion time. Data were obtained from the 2016 London Marathon participants. Participants completed the Athlete Sleep Screening Questionnaire (ASSQ) along with a brief survey capturing individual characteristics and lifestyle factors. Sleep health focused on the ASSQ sleep difficulty score (SDS) and its components. Linear regression computed relations among sleep, individual, lifestyle, and marathon variables. The analytic sample (N = 943) was mostly male (64.5%) and young adults (66.5%). A total of 23.5% of the sample reported sleep difficulties (SDS ≥ 8) at a severity warranting follow-up with a trained sleep provider. Middle-aged adults generally reported significantly worse sleep health characteristics, relative to young adults, except young adults reported significantly longer sleep onset latency (SOL). Sleep tracker users reported worse sleep satisfaction. Pre-bedtime electronic device use was associated with longer SOL and longer marathon completion time, while increasing SOL was also associated with longer marathon completion. Our results suggest a deleterious influence of pre-bedtime electronic device use and sleep tracker use on sleep health in marathoners. Orthosomnia may be a relevant factor in the relationship between sleep tracking and sleep health for marathoners

Fabrication of cell container arrays with overlaid surface topographies

This paper presents cell culture substrates in the form of microcontainer arrays with overlaid surface topographies, and a technology for their fabrication. The new fabrication technology is based on microscale thermoforming of thin polymer films whose surfaces are topographically prepatterned on a micro- or nanoscale. For microthermoforming, we apply a new process on the basis of temporary back moulding of polymer films and use the novel concept of a perforated-sheet-like mould. Thermal micro- or nanoimprinting is applied for prepatterning. The novel cell container arrays are fabricated from polylactic acid (PLA) films. The thin-walled microcontainer structures have the shape of a spherical calotte merging into a hexagonal shape at their upper circumferential edges. In the arrays, the cell containers are arranged densely packed in honeycomb fashion. The inner surfaces of the highly curved container walls are provided with various topographical micro- and nanopatterns. For a first validation of the microcontainer arrays as in vitro cell culture substrates, C2C12 mouse premyoblasts are cultured in containers with microgrooved surfaces and shown to align along the grooves in the three-dimensional film substrates. In future stem-cell-biological and tissue engineering applications, microcontainers fabricated using the proposed technology may act as geometrically defined artificial microenvironments or niches

Calibration of X-ray imaging devices for accurate intensity measurement

National Security Technologies (NSTec) has developed calibration procedures for X-ray imaging systems. The X-ray sources that are used for calibration are both diode type and diode/fluorescer combinations. Calibrating the X-ray detectors is key to accurate calibration of the X-ray sources. Both energy dispersive detectors and photodiodes measuring total flux were used. We have developed calibration techniques for the detectors using radioactive sources that are traceable to the National Institute of Standards and Technology (NIST). The German synchrotron at Physikalische Technische Bundestalt (PTB) is used to calibrate silicon photodiodes over the energy range from 50 eV to 60 keV. The measurements on X-ray cameras made using the NSTec X-ray sources have included quantum efficiency averaged over all pixels, camera counts per photon per pixel, and response variation across the sensor. The instrumentation required to accomplish the calibrations is described. X-ray energies ranged from 720 eV to 22.7 keV. The X-ray sources produce narrow energy bands, allowing us to determine the properties as a function of X-ray energy. The calibrations were done for several types of imaging devices. There were back illuminated and front illuminated CCD (charge coupled device) sensors, and a CID (charge injection device) type camera. The CCD and CID camera types differ significantly in some of their properties that affect the accuracy of X-ray intensity measurements. All cameras discussed here are silicon based. The measurements of quantum efficiency variation with X-ray energy are compared to models for the sensor structure. Cameras that are not back-thinned are compared to those that are

Chromosome 3 Anomalies Investigated by Genome Wide SNP Analysis of Benign, Low Malignant Potential and Low Grade Ovarian Serous Tumours

Ovarian carcinomas exhibit extensive heterogeneity, and their etiology remains unknown. Histological and genetic evidence has led to the proposal that low grade ovarian serous carcinomas (LGOSC) have a different etiology than high grade carcinomas (HGOSC), arising from serous tumours of low malignant potential (LMP). Common regions of chromosome (chr) 3 loss have been observed in all types of serous ovarian tumours, including benign, suggesting that these regions contain genes important in the development of all ovarian serous carcinomas. A high-density genome-wide genotyping bead array technology, which assayed >600,000 markers, was applied to a panel of serous benign and LMP tumours and a small set of LGOSC, to characterize somatic events associated with the most indolent forms of ovarian disease. The genomic patterns inferred were related to TP53, KRAS and BRAF mutations. An increasing frequency of genomic anomalies was observed with pathology of disease: 3/22 (13.6%) benign cases, 40/53 (75.5%) LMP cases and 10/11 (90.9%) LGOSC cases. Low frequencies of chr3 anomalies occurred in all tumour types. Runs of homozygosity were most commonly observed on chr3, with the 3p12-p11 candidate tumour suppressor region the most frequently homozygous region in the genome. An LMP harboured a homozygous deletion on chr6 which created a GOPC-ROS1 fusion gene, previously reported as oncogenic in other cancer types. Somatic TP53, KRAS and BRAF mutations were not observed in benign tumours. KRAS-mutation positive LMP cases displayed significantly more chromosomal aberrations than BRAF-mutation positive or KRAS and BRAF mutation negative cases. Gain of 12p, which harbours the KRAS gene, was particularly evident. A pathology review reclassified all TP53-mutation positive LGOSC cases, some of which acquired a HGOSC status. Taken together, our results support the view that LGOSC could arise from serous benign and LMP tumours, but does not exclude the possibility that HGOSC may derive from LMP tumours