Ultrasound-guided hepatic portal vein injection is not a reproducible technique for delivery of cell therapies to the liver in mice.

AIMS: Our aim was to determine if ultrasound-guided HPV injection in mice would provide reproducible and reliable results, as is currently obtained via open laparotomy techniques, and offer a surgical refinement to emulate islet transplantation in humans. METHODS: Fluorescent-polymer microparticles (20 µm) were injected (27G-needle) into the HPV via open laparotomy (n = 4) or under ultrasound-guidance (n = 4) using an MX550D-transducer with a Vevo3100-scanner (FUJIFILM VisualSonics, Inc.). Mice were culled 24-h post injection; organs were frozen, step sectioned (10 µm-slices) and 10 sections/mouse (50 µm-spacing) were quantified for microparticles in the liver and other organs by fluorescent microscopy. RESULTS: Murine HPV injection, via open laparotomy-route, resulted in widespread distribution of microparticles in the liver, lungs and spleen; ultrasound-guided injection resulted in reduced microparticle delivery (p < 0.0001) and microparticle clustering in distinct areas of the liver at the site of needle penetration, with very few/no microparticles being seen in lung and spleen tissues, hypothesised to be due to flow into the body cavity: liver median (interquartile range) 4.15 (0.00-4.15) versus 0.00 (0.00-0.00) particle-count mm-2 , respectively. CONCLUSIONS: Ultrasound-guided injection results in microparticle clustering in the liver, with an overall reduction in microparticle number when compared to open laparotomy HPV injection, and high variability in microparticle-counts detected between mice. Ultrasound-guided injection is not currently a technique that can replace open laparotomy HPV of islet transplantation in mice.

Performance evaluation of a non-invasive one-step multiplex RT-qPCR assay for detection of SARS-CoV-2 direct from saliva.

Polymerase chain reaction (PCR) has proven to be the gold-standard for SARS-CoV-2 detection in clinical settings. The most common approaches rely on nasopharyngeal specimens obtained from swabs, followed by RNA extraction, reverse transcription and quantitative PCR. Although swab-based PCR is sensitive, swabbing is invasive and unpleasant to administer, reducing patient compliance for regular testing and resulting in an increased risk of improper sampling. To overcome these obstacles, we developed a non-invasive one-step RT-qPCR assay performed directly on saliva specimens. The University of Nottingham Asymptomatic Testing Service protocol simplifies sample collection and bypasses the need for RNA extraction, or additives, thus helping to encourage more regular testing and reducing processing time and costs. We have evaluated the assay against the performance criteria specified by the UK regulatory bodies and attained accreditation (BS EN ISO/IEC 17,025:2017) for SARS-CoV-2 diagnostic testing by the United Kingdom Accreditation Service. We observed a sensitivity of 1 viral copy per microlitre of saliva, and demonstrated a concordance of > 99.4% between our results and those of other accredited testing facilities. We concluded that saliva is a stable medium that allows for a highly precise, repeatable, and robust testing method.

Direct RT-qPCR Assay for the Detection of SARS-CoV-2 in Saliva Samples.

Since mid-2020 there have been complexities and difficulties in the standardisation and administration of nasopharyngeal swabs. Coupled with the variable and/or poor accuracy of lateral flow devices, this has led to increased societal 'testing fatigue' and reduced confidence in test results. Consequently, asymptomatic individuals have developed reluctance towards repeat testing, which remains the best way to monitor COVID-19 cases in the wider population. On the other hand, saliva-based PCR, a non-invasive, highly sensitive, and accurate test suitable for everyone, is gaining momentum as a straightforward and reliable means of detecting SARS-CoV-2 in symptomatic and asymptomatic individuals. Here, we provide an itemised list of the equipment and reagents involved in the process of sample submission, inactivation and analysis, as well as a detailed description of how each of these steps is performed.

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies.

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive ('additive') top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Photoresponsive Hydrogels with Photoswitchable Mechanical Properties Allow Time-Resolved Analysis of Cellular Responses to Matrix Stiffening.

As cell function and phenotype can be directed by the mechanical characteristics of the surrounding matrix, hydrogels have become important platforms for cell culture systems, with properties that can be tuned by external stimuli, such as divalent cations, enzymatic treatment, and pH. However, many of these stimuli can directly affect cell behavior, making it difficult to distinguish purely mechanical signaling events. This study reports on the development of a hydrogel that incorporates photoswitchable cross-linkers, which can reversibly alter their stiffness upon irradiation with the appropriate wavelength of light. Furthermore, this study reports the response of bone-marrow-derived mesenchymal stem cells (MSCs) on these hydrogels that were stiffened systematically by irradiation with blue light. The substrates were shown to be noncytotoxic, and crucially MSCs were not affected by blue-light exposure. Time-resolved analysis of cell morphology showed characteristic cell spreading and increased aspect ratios in response to greater substrate stiffness. This hydrogel provides a platform to study mechanosignaling in cells responding to dynamic changes in stiffness, offering a new way to study mechanotransduction signaling pathways and biological processes, with implicit changes to tissue mechanics, such as development, ageing, and fibrosis.