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INTESTINAL Publication-derived

Rotavirus Infection Intestinal Organoids for Drug Screening

Full SOP with Academic or a plan

Source Yin et al., 2015 · Erasmus MC-University Medical Center, Rotterdam, The Netherlands · 10.1016/j.antiviral.2015.09.010

Authors: Yuebang Yin, Marcel Bijvelds, Wen Dang, Lei Xu, Annemiek A. van der Eijk, Karen Knipping, Nesrin Tuysuz, Johanna F. Dekkers, Yijin Wang, Jeroen de Jonge, Dave Sprengers, Luc J.W. van der Laan, Jeffrey M. Beekman, Derk ten Berge, Herold J. Metselaar, Hugo de Jonge, Marion P.G. Koopmans, Maikel P. Peppelenbosch, Qiuwei Pan Duration: 7 days 11 phases Cell source: Human intestinal epithelial cells from biopsy/surgical resection and Mouse intestinal epithelial cells from crypts

Abstract

This protocol describes the establishment of three-dimensional primary intestinal organoid cultures from human and mouse tissues and their use as a model system for rotavirus infection and antiviral therapy testing. Human organoids demonstrate high permissiveness to rotavirus, supporting both laboratory-adapted (SA11) and patient-derived strains, with robust production of infectious viral particles. The system enables rapid assessment of individual responsiveness to antiviral agents including interferon-alpha and ribavirin.

Cell source
Human intestinal epithelial cells from biopsy/surgical resection and Mouse intestinal epithelial cells from crypts
Application
Disease modeling, Antiviral drug screening

Protocol overview

44 steps across 11 phases

Phase 1 · Primary Mouse Intestinal Organoid Culture Days 0-5 (initial culture), Days 5-6 (passaging)
  1. a Mouse tissue harvesting and preparation
  2. b Crypt isolation with EDTA
  3. c Crypt collection and suspension
  4. d Matrigel embedding and solidification
  5. e Organoid culture medium addition and maintenance
  6. f Organoid passaging
Phase 2 · Primary Human Intestinal Organoid Culture Days 0-7 (initial culture), Days 7+ (passaging)
  1. a Human tissue collection and preparation
  2. b Tissue washing and EDTA treatment
  3. c Crypt release and collection
  4. d Crypt resuspension and Matrigel embedding
  5. e Matrigel embedding and solidification
  6. f Organoid culture with enhanced medium
  7. g Organoid passaging
Phase 3 · SA11 Rotavirus Inoculation into Organoids Day 1 (infection protocol)
  1. a Virus activation with trypsin
  2. b Organoid inoculation
  3. c Removal of free virus
  4. d Re-embedding and culture
Phase 4 · Patient-Derived Rotavirus Inoculation Day 1 (infection protocol)
  1. a Stool sample preparation
  2. b Inoculation of human organoids
Phase 5 · Viral Production Assay Days 1-3 (sampling period)
  1. a Culture medium collection
  2. b Infectivity confirmation with secondary infection
Phase 6 · Antiviral Treatment: Interferon-Alpha (IFN-α) Days 1-3 (treatment and analysis)
  1. a IFN-α pretreatment
  2. b Viral inoculation and continued treatment
  3. c RNA analysis and quantification
Phase 7 · Antiviral Treatment: Ribavirin Days 1-3 (treatment and analysis)
  1. a Ribavirin treatment
  2. b Dose-dependent analysis
  3. c RNA extraction and qRT-PCR analysis
Phase 8 · Neutralization Assay with Monoclonal Antibody Day 1 (antibody incubation and infection)
  1. a Virus activation and antibody incubation
  2. b Inoculation of MAb-treated virus
  3. c Viral RNA quantification
Phase 9 · Immunohistochemistry (IHC) for Viral Protein Detection Days 1-2 (sample preparation and staining)
  1. a Organoid fixation and preservation
  2. b Organoid dehydration and processing
  3. c Section preparation
  4. d Deparaffinization and antigen retrieval
  5. e Blocking and primary antibody incubation
  6. f Secondary antibody incubation and counterstaining
  7. g Visualization
Phase 10 · qRT-PCR for Rotavirus RNA and ISG Detection Days 1-3 (RNA extraction and analysis)
  1. a Total RNA extraction
  2. b cDNA synthesis
  3. c qRT-PCR reaction setup and cycling conditions
  4. d Gene expression analysis and normalization
Phase 11 · Rotavirus Genome Copy Number Quantification Days 0-1 (standard curve generation and analysis)
  1. a Standard plasmid preparation
  2. b Standard curve generation
  3. c Copy number calculation

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Attribution

This SOP was written by Organthis based on the published method in Yin et al., 2015. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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