viral infection of human intestinal organoid monolayers for live fluorescence imaging

Imagerie calcique de monocouches d’organoïdes intestinaux humains infectés par un virus

Calcium is a widely conserved second messenger that plays a critical role in regulating cellular physiology1. Given its strong charge, small size, and high solubility in physiological conditions, calcium is an ideal manipulator of protein conformation. This makes calcium a powerful means to transduce electrochemical signals into enzymatic, transcriptional, or post-transcriptional alterations. The strict calcium concentration gradients across the endoplasmic reticulum (ER) and plasma membranes create a high driving force that allows for rapid changes in cytosolic calcium concentration. Multiple mechanisms, including both buffering and active transport, tightly maintain this gradient. While necessary for normal cellular functions, this maintenance is energetically expensive, making it particularly susceptible in states of stress 2.
As such, dysregulation of calcium within the cytosol is a near-universal signal of many kinds of cellular stress. Metabolic disturbances, toxins, pathogens, mechanical damage, and genetic perturbations can all disrupt calcium signaling. Regardless of the stimulus, on a whole-cell level, sustained, uncontrolled rises in cytosolic calcium can promote apoptosis and&#160;eventually necrosis3,4. Alterations in cytosolic calcium levels of lower amplitude or higher frequency, however, have varying effects2. Likewise, the outcomes of calcium fluctuations may differ based on the spatial microdomain in which they occur5. Monitoring calcium levels can&#160;therefore offer insight into dynamic signaling processes, but this requires sampling with relatively high temporal and spatial resolution.
Genetically encoded calcium indicators (GECIs) are powerful tools for continuous sampling in live-cell systems6. Some of the most widely used GECIs are GFP-based calcium-responsive fluorescent proteins known as GCaMPs7. The canonical GCaMP is a fusion of three distinct protein domains: a circularly permuted GFP (cpGFP), calmodulin, and M136. The calmodulin domain undergoes a conformation change upon binding calcium, allowing its interaction with M13. The calmodulin-M13 interaction induces a conformational change in the cpGFP that increases its fluorescent emission upon excitation. As such, an increase in calcium concentration correlates with an increase in GCaMP fluorescence intensity. These sensors can be cytosolic or targeted to specific organelles8.
Similar to most tissues, calcium regulates a variety of functions within the gastrointestinal epithelium. The intestinal epithelium is integral for nutrient and fluid absorption but also must form a tight barrier and immune interface to avoid pathogen invasion or toxic insults. Calcium-dependent pathways influence nearly all of these vital functions9,10,11. However, calcium signaling within the intestinal epithelium remains an underexplored frontier with promising potential as a therapeutic target. While monitoring calcium dynamics within the intestinal epithelium in vivo continues to present challenges, human intestinal organoids (HIOs) offer an adaptable ex vivo system for experimentation12. HIOs are 3-dimensional (3D) spheroids derived from human intestinal stem cells and, upon differentiation, recapitulate much of the cellular diversity of the native intestinal epithelium12.
This protocol describes comprehensive methods to engineer HIOs that express GECIs and then prepare engineered HIOs as monolayers for live-cell calcium imaging. It offers viral infection as an example of a pathologic manipulation that disrupts calcium signaling and provides an analytic approach to quantify these changes.

Pleins feux sur l’auteur : Étude de la perturbation virale de la signalisation épithéliale intestinale – Perspectives de recherche et orientations futures 

Imagerie calcique en direct de monocouches organoïdes intestinaux humains infectés par un virus à l’aide d’indicateurs de calcium codés génétiquement

Through our work, we aim to understand how viruses disrupt signaling within the intestinal epithelium. We are particularly interested in understanding the ways in which virus-infected cells dysregulate neighboring uninfected cells to contribute to pathogenesis. We've recently discovered that rotavirus-infected cells release pulses of ADP, which dysregulates neighboring uninfected cells and facilitates viral spread and exacerbates disease.While calcium imaging is very popular in fields like neuroscience, it has not been widely used to study epithelial biology or virology. This protocol offers an approach to adapt existing technologies and model systems to study calcium signaling and live virus-infected epithelial tissue. By engineering organoids to express genetically encoded calcium indicators, our approach allows for a consistent, reproducible long-term imaging in a model system that recapitulates the cellular diversity of the human intestinal epithelium.We aim to understand the effects of virus-induced paracrine purinergic signaling for both the host and the virus. Our future work will also determine whether this type of signaling is broadly conserved across other viruses and whether the effects are similar.

Watch this Scientific Journal Video about Viral Infection of Human Intestinal Organoid Monolayers for Live Fluorescence Imaging at JoVE.com

Viral Infection of Human Intestinal Organoid Monolayers for Live Fluorescence Imaging  

Infection virale de monocouches organoïdes intestinaux humains pour l’imagerie par fluorescence en direct

Commencez par préparer l’inoculum du virus. Activez le stock de rotavirus en ajoutant 10 microgrammes par microlitre de trypsine de Worthington et en incubant à 37 degrés Celsius pendant une heure. Ensuite, diluez 50 microlitres de la souche virale activée avec 50 microlitres de CMGF-Ensuite, remplacez le milieu des monocouches HIO par 100 microlitres d’inoculum viral pour infecter les monocouches.Dans le cas d’une infection à rotavirus, faites une entaille unique sur toute la longueur de la monocouche à l’aide d’une aiguille de calibre 25. Incubez les assiettes à 37 degrés Celsius pendant deux heures. Après l’incubation, retirez l’inoculum et lavez les monocouches une fois avec 1 X PBS.Ajoutez ensuite 100 microlitres de milieu de différenciation sans rouge phénolique. Placez la lame dans un incubateur à 37 degrés Celsius jusqu’à ce qu’elle soit prête à être photographiée.

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Research

JoVE Journal

Immunology and Infection

Viral Infection of Human Intestinal Organoid Monolayers for Live Fluorescence Imaging

106 vues.  Baylor College of Medicine. Commencez par préparer l’inoculum du virus. Activez le stock de rotavirus en ajoutant 10 microgrammes par microlitre de trypsine de Worthington et en incubant à 37 degrés Celsius pendant une heure. Ensuite, diluez 50 microlitres de la souche virale activée avec 50 microlitres de CMGF-Ensuite, remplacez le milieu des monocouches HIO par 100 microlitres d’inoculum viral pour infecter les monocouches.Dans le cas d’une infection à rotavirus, faites une entaille unique sur toute ...

Vidéo: Viral Infection of Human Intestinal Organoid Monolayers for Live Fluorescence Imaging  

Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory activity, actin dynamics, inflammatory responses, stem cell proliferation, and many other uncharacterized cellular functions. As such, mapping calcium signaling dynamics within the intestinal epithelium can provide insight into homeostatic cellular processes and unveil unique responses to various stimuli. Human intestinal organoids (HIOs) are a high-throughput, human-derived model to study the intestinal epithelium and thus represent a useful system to investigate calcium dynamics. This paper describes a protocol to stably transduce HIOs with genetically encoded calcium indicators (GECIs), perform live fluorescence microscopy, and analyze imaging data to meaningfully characterize calcium signals. As a representative example, 3-dimensional HIOs were transduced with lentivirus to stably express GCaMP6s, a green fluorescent protein-based cytosolic GECI. The engineered HIOs were then dispersed into a single-cell suspension and seeded as monolayers. After differentiation, the HIO monolayers were infected with rotavirus and/or treated with drugs known to stimulate a calcium response. An epifluorescence microscope fitted with a temperature-controlled, humidified live-imaging chamber allowed for long-term imaging of infected or drug-treated monolayers. Following imaging, acquired images were analyzed using the freely available analysis software, ImageJ. Overall, this work establishes an adaptable pipeline for characterizing cellular signaling in HIOs.

This protocol describes an approach for performing calcium imaging in virus-infected human intestinal organoids and offers an approach to analysis.

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory ...