Analysis of enterovirus infection is difficult in animals because they express different virus receptors than humans, and static cell culture systems do not reproduce the physical complexity of the ...human intestinal epithelium. Here, using coxsackievirus B1 (CVB1) as a prototype enterovirus strain, we demonstrate that human enterovirus infection, replication and infectious virus production can be analyzed in vitro in a human Gut-on-a-Chip microfluidic device that supports culture of highly differentiated human villus intestinal epithelium under conditions of fluid flow and peristalsis-like motions. When CVB1 was introduced into the epithelium-lined intestinal lumen of the device, virions entered the epithelium, replicated inside the cells producing detectable cytopathic effects (CPEs), and both infectious virions and inflammatory cytokines were released in a polarized manner from the cell apex, as they could be detected in the effluent from the epithelial microchannel. When the virus was introduced via a basal route of infection (by inoculating virus into fluid flowing through a parallel lower 'vascular' channel separated from the epithelial channel by a porous membrane), significantly lower viral titers, decreased CPEs, and delayed caspase-3 activation were observed; however, cytokines continued to be secreted apically. The presence of continuous fluid flow through the epithelial lumen also resulted in production of a gradient of CPEs consistent with the flow direction. Thus, the human Gut-on-a-Chip may provide a suitable in vitro model for enteric virus infection and for investigating mechanisms of enterovirus pathogenesis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
RNA-sequencing (RNA-seq) measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. In contrast, in situ hybridization provides the location of ...gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq, our method enriches for context-specific transcripts over housekeeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d.
Abstract The performance of biomaterials-based therapies can be hindered by complications associated with surgical implant, motivating the development of materials systems that allow minimally ...invasive introduction into the host. In this study, we created cell-adhesive and degradable gelatin scaffolds that could be injected through a conventional needle while maintaining a predefined geometry and architecture. These scaffolds supported attachment, proliferation, and survival of cells in vitro and could be degraded by recombinant matrix metalloproteinase-2 and -9. Prefabricated gelatin cryogels rapidly resumed their original shape when injected subcutaneously into mice and elicited only a minor host response following injection. Controlled release of granulocyte-macrophage colony-stimulating factor from gelatin cryogels resulted in complete infiltration of the scaffold by immune cells and promoted matrix metalloproteinase production leading to cell-mediated degradation of the cryogel matrix. These findings suggest that gelatin cryogels could serve as a cell-responsive platform for biomaterial-based therapy.
Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe ...fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.
Pluripotent stem cells (PSCs) are capable of dynamic interconversion between distinct substates; however, the regulatory circuits specifying these states and enabling transitions between them are not ...well understood. Here we set out to characterize transcriptional heterogeneity in mouse PSCs by single-cell expression profiling under different chemical and genetic perturbations. Signalling factors and developmental regulators show highly variable expression, with expression states for some variable genes heritable through multiple cell divisions. Expression variability and population heterogeneity can be influenced by perturbation of signalling pathways and chromatin regulators. Notably, either removal of mature microRNAs or pharmacological blockage of signalling pathways drives PSCs into a low-noise ground state characterized by a reconfigured pluripotency network, enhanced self-renewal and a distinct chromatin state, an effect mediated by opposing microRNA families acting on the Myc/Lin28/let-7 axis. These data provide insight into the nature of transcriptional heterogeneity in PSCs.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Here we describe the development of a human lung 'small airway-on-a-chip' containing a differentiated, mucociliary bronchiolar epithelium and an underlying microvascular endothelium that experiences ...fluid flow, which allows for analysis of organ-level lung pathophysiology in vitro. Exposure of the epithelium to interleukin-13 (IL-13) reconstituted the goblet cell hyperplasia, cytokine hypersecretion and decreased ciliary function of asthmatics. Small airway chips lined with epithelial cells from individuals with chronic obstructive pulmonary disease recapitulated features of the disease such as selective cytokine hypersecretion, increased neutrophil recruitment and clinical exacerbation by exposure to viral and bacterial infections. With this robust in vitro method for modeling human lung inflammatory disorders, it is possible to detect synergistic effects of lung endothelium and epithelium on cytokine secretion, identify new biomarkers of disease exacerbation and measure responses to anti-inflammatory compounds that inhibit cytokine-induced recruitment of circulating neutrophils under flow.
Kidney organoids derived from human pluripotent stem cells have glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here we report an in vitro method ...for culturing kidney organoids under flow on millifluidic chips, which expands their endogenous pool of endothelial progenitor cells and generates vascular networks with perfusable lumens surrounded by mural cells. We found that vascularized kidney organoids cultured under flow had more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression compared with that in static controls. Glomerular vascular development progressed through intermediate stages akin to those involved in the embryonic mammalian kidney's formation of capillary loops abutting foot processes. The association of vessels with these compartments was reduced after disruption of the endogenous VEGF gradient. The ability to induce substantial vascularization and morphological maturation of kidney organoids in vitro under flow opens new avenues for studies of kidney development, disease, and regeneration.
An integrated, low-cost, sample-to-answer, CRISPR-based diagnostic detects SARS-CoV-2 and variants from unprocessed saliva.
The COVID-19 pandemic highlights the need for diagnostics that can be ...rapidly adapted and deployed in a variety of settings. Several SARS-CoV-2 variants have shown worrisome effects on vaccine and treatment efficacy, but no current point-of-care (POC) testing modality allows their specific identification. We have developed miSHERLOCK, a low-cost, CRISPR-based POC diagnostic platform that takes unprocessed patient saliva; extracts, purifies, and concentrates viral RNA; performs amplification and detection reactions; and provides fluorescent visual output with only three user actions and 1 hour from sample input to answer out. miSHERLOCK achieves highly sensitive multiplexed detection of SARS-CoV-2 and mutations associated with variants B.1.1.7, B.1.351, and P.1. Our modular system enables easy exchange of assays to address diverse user needs and can be rapidly reconfigured to detect different viruses and variants of concern. An adjunctive smartphone application enables output quantification, automated interpretation, and the possibility of remote, distributed result reporting.
Smoking represents a major risk factor for chronic obstructive pulmonary disease (COPD), but it is difficult to characterize smoke-induced injury responses under physiological breathing conditions in ...humans due to patient-to-patient variability. Here, we show that a small airway-on-a-chip device lined by living human bronchiolar epithelium from normal or COPD patients can be connected to an instrument that “breathes” whole cigarette smoke in and out of the chips to study smoke-induced pathophysiology in vitro. This technology enables true matched comparisons of biological responses by culturing cells from the same individual with or without smoke exposure. These studies led to identification of ciliary micropathologies, COPD-specific molecular signatures, and epithelial responses to smoke generated by electronic cigarettes. The smoking airway-on-a-chip represents a tool to study normal and disease-specific responses of the human lung to inhaled smoke across molecular, cellular and tissue-level responses in an organ-relevant context.
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•Smoking lung airway chip recapitulated clinical oxidative stress molecular profiles•New smoke-induced ciliary micropathologies were identified•This technology supported study of potential toxic effects of electronic cigarettes•COPD-specific responses were reproduced in vitro and novel biomarkers were identified
Benam et al. describe a microengineered in vitro model system that permits analysis of the effects of whole smoke, from both conventional tobacco and electronic cigarettes, delivered under physiologically relevant flow conditions that mimic breathing on the pathophysiology of differentiated human mucociliated bronchiolar epithelium cultured in a microfluidic small airway-on-a-chip.
Exposure of lung tissues to cigarette smoke is a major cause of human disease and death worldwide. Unfortunately, adequate model systems that can reliably recapitulate disease biogenesis in vitro, ...including exposure of the human lung airway to fresh whole cigarette smoke (WCS) under physiological breathing airflow, are lacking. This protocol extension builds upon, and can be used with, our earlier protocol for microfabrication of human organs-on-chips. Here, we describe the engineering, assembly and operation of a microfluidically coupled, multi-compartment platform that bidirectionally 'breathes' WCS through microchannels of a human lung small airway microfluidic culture device, mimicking how lung cells may experience smoke in vivo. Several WCS-exposure systems have been developed, but they introduce smoke directly from above the cell cultures, rather than tangentially as naturally occurs in the lung due to lateral airflow. We detail the development of an organ chip-compatible microrespirator and a smoke machine to simulate breathing behavior and smoking topography parameters such as puff time, inter-puff interval and puffs per cigarette. Detailed design files, assembly instructions and control software are provided. This novel platform can be fabricated and assembled in days and can be used repeatedly. Moderate to advanced engineering and programming skills are required to successfully implement this protocol. When coupled with the small airway chip, this protocol can enable prediction of patient-specific biological responses in a matched-comparative manner. We also demonstrate how to adapt the protocol to expose living ciliated airway epithelial cells to smoke generated by electronic cigarettes (e-cigarettes) on-chip.
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