Development of a skin-on-a-chip platform for the study of wound healing

Wound healing abnormalities and non-healing chronic wounds are a major clinical problem, primarily affecting diabetic and elderly patients. Wound management associated costs resulted in a £5.3 billion financial burden to the NHS between 2012 and 2013. These serious medical states are being recognised as mortal disease, with the fatality rates often higher than those of common cancers. Half of all patients undergoing chronic-wound associated amputation related to diabetes are expected to die within 5 years. The current chronic wound treatments are inadequate, and more sophisticated models are needed to advance this field, leading to better therapies. The aim of this work was to design, manufacture, and test a microfluidic device that would address the need for a physiologically accurate model of wound healing with improved assay lifespan, when compared to the classical static models. Here we present a new microfluidic device optimised for maintaining human skin samples for prolonged period of time, and wound healing analysis. Briefly, full thickness human skin explant samples were cultured on custom-made microfluidic devices, and in static culture. The skin samples were collected at the end of the culture period, fixed, sectioned and either stained using fluorescent TUNEL assay to analyse the tissue apoptosis, or using IHC, for K6 and K14 to study tissue architecture. Wound healing outcome was measured using wound samples wholemount-stained for K1 and K14. FACS analysis was performed on digested samples to study the immunological profile in the cultured samples. The final version of the skin-on-a-chip device (V4.0) was found to be successful at prolonging tissue survival. After the seven day culture period, four-fold decrease in the epidermal apoptotic cell death (1.4% apoptosis for the on-chip sample vs. 6.1% for the static control), and two-fold decrease in the dermal apoptosis (4.1% cell death in the flow-chip samples vs. 8.3% in the static controls) were observed. Day 7 samples maintained on the V4.0 device significantly outperformed the static control samples (p = 0.007). Furthermore, the average dermal cell death for the control samples collected on day 14 was 38.1% whilst the on-chip samples exhibited dermal cell death averaging 8.3%. V4.0 samples contain significantly less apoptotic cells in the dermal section when compared to the static controls on day 14 (p = 0.0433675). The improved tissue viability makes the model more suitable for prolonged culture experiments. Next, it was observed that the wound area is reducing in size over the period of seven days, in both cases of the V4.0 samples and the control samples. The on-chip samples yield reduced wound perimeter when compared to the static controls from the same day. the culture method has a very significant influence on the wound size (f(1) = 75.684, p=5.61x10-6). Interestingly, the same analysis showed that the culture method does have a greater impact on the wound closure that the assay day (f(2) = 24.615, p = 0.012). The on-chip samples produced a significantly different smaller wounds on day seven of the assay than the control static culture samples (p = 0.012). In addition, the overall theme seen from the FACS data demonstrated that the wounded skin samples cultured on the V4.0 microfluidic devices yielded higher levels of immune cells than the static control samples collected on the corresponding days. Overall, The V4.0 device allowed for an increased number of cells to be collected on day seven in every single marker group, apart from in the CD56+ CD3+ group, where the levels dropped more in the V4.0 samples than in the control samples. The same relationship was noted on day three. This indicates that maintaining samples on the V4.0 device helps to improve the immune cell retention, making the microfluidic model’s immune microenvironment more comparable to the real human skin microenvironment

How to study basement membrane stiffness as a biophysical trigger in prostate cancer and other age-related pathologies or metabolic diseases

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e.g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by ((sodium dodecyl sulfate polyacrylamide gel electrophoresis)) (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed

PySDM v1 : particle-based cloud modeling package for warm-rain microphysics and aqueous chemistry

PySDM is an open-source Python package for simulating the dynamics of particles undergoing condensational and collisional growth, interacting with a fluid flow and subject to chemical composition changes. It is intended to serve as a building block for process-level as well as computational-fluid-dynamics simulation systems involving representation of a continuous phase (air) and a dispersed phase (aerosol), with PySDM being responsible for representation of the dispersed phase. The PySDM package core is a Pythonic high-performance implementation of the Super-Droplet Method (SDM) Monte-Carlo algorithm for representing collisional growth, hence the name. PySDM has two alternative parallel number-crunching backends available: multi-threaded CPU backend based on Numba and GPU-resident backend built on top of ThrustRTC. The usage examples are built on top of four simple atmospheric cloud modelling frameworks: box, adiabatic parcel, single-column and 2D prescribed flow kinematic models. In addition, the package ships with tutorial code depicting how PySDM can be used from Julia and Matlab

Adenosine Receptor Agonist HE-NECA Enhances Antithrombotic Activities of Cangrelor and Prasugrel in vivo by Decreasing of Fibrinogen Density in Thrombus

Blood platelets’ adenosine receptors (AR) are considered to be a new target for the anti-platelet therapy. This idea is based on in vitro studies which show that signaling mediated by these receptors leads to a decreased platelet response to activating stimuli. In vivo evidence for the antithrombotic activity of AR agonists published to date were limited, however, to the usage of relatively high doses given in bolus. The present study was aimed at verifying if these substances used in lower doses in combination with inhibitors of P2Y12 could serve as components of dual anti-platelet therapy. We have found that a selective A2A agonist 2-hexynyl-5’-N-ethylcarboxamidoadenosine (HE-NECA) improved the anti-thrombotic properties of either cangrelor or prasugrel in the model of ferric chloride-induced experimental thrombosis in mice. Importantly, HE-NECA was effective not only when applied in bolus as other AR agonists in the up-to-date published studies, but also when given chronically. In vitro thrombus formation under flow conditions revealed that HE-NECA enhanced the ability of P2Y12 inhibitors to decrease fibrinogen content in thrombi, possibly resulting in their lower stability. Adenosine receptor agonists possess a certain hypotensive effect and an ability to increase the blood–brain barrier permeability. Therefore, the effects of anti-thrombotic doses of HE-NECA on blood pressure and the blood–brain barrier permeability in mice were tested. HE-NECA applied in bolus caused a significant hypotension in mice, but the effect was much lower when the substance was given in doses corresponding to that obtained by chronic administration. At the same time, no significant effect of HE-NECA was observed on the blood–brain barrier. We conclude that chronic administration of the A2A agonist can be considered a potential component of a dual antithrombotic therapy. However, due to the hypotensive effect of the substances, dosage and administration must be elaborated to minimize the side-effects. The total number of animals used in the experiments was 146

PySDM

Pythonic particle-based (super-droplet) warm-rain/aqueous-chemistry cloud microphysics package with box, parcel & 1D/2D prescribed-flow examples in Python, Julia and Matla