Name: Viral Infection of Human Intestinal Organoid Monolayers for Live Fluorescence Imaging
Uploaded: 2024-01-19T14:05:00
Description: 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

Kalsiumavbildning av virusinfiserte humane tarmorganoide monolag

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.

Forfatter Spotlight: Undersøkelse av viral forstyrrelse av tarmepitelsignalering – forskningsinnsikt og fremtidige retninger 

Levende kalsiumavbildning av virusinfiserte humane intestinale organoidmonolag ved bruk av genetisk kodede kalsiumindikatorer

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  

Viral infeksjon av humane intestinale organoide monolayers for levende fluorescensavbildning

Begynn med å forberede virusinokulumet. Aktiver rotavirusbestanden ved å tilsette 10 mikrogram per mikroliter Worthingtons trypsin og inkubere ved 37 grader Celsius i en time. Fortynn deretter 50 mikroliter av den aktiverte virusbestanden med 50 mikroliter CMGF-Deretter erstatter du mediet fra HIO-monolagene med 100 mikroliter virusinokulum for å infisere monolagene.For rotavirusinfeksjon, lag en enkelt poengsum over monolagets lengde ved hjelp av en 25-gauge nål. Inkuber platene ved 37 grader Celsius i to timer. Etter inkubering, fjern inokulumet og vask monolagene en gang med 1 X PBS.Deretter tilsettes 100 mikroliter fenolfritt differensieringsmedium. Plasser lysbildet i en 37-graders Celsius-inkubator til du er klar til å avbilde.

Research

JoVE Journal

Immunology and Infection

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 ...