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INTESTINAL Publication-derived

Rotavirus Infection Small Intestinal Models for Disease Modelling

Full SOP with Academic or a plan

Source Saxena et al., 2016 · Baylor College of Medicine · 10.1128/jvi.01930-15

Authors: Kapil Saxena, Sarah E. Blutt, Khalil Ettayebi, Xi-Lei Zeng, James R. Broughman, Sue E. Crawford, Umesh C. Karandikar, Narayan P. Sastri, Margaret E. Conner, Antone R. Opekun, David Y. Graham, Waqar Qureshi, Vadim Sherman, Jennifer Foulke-Abel, Julie In, Olga Kovbasnjuk, Nicholas C. Zachos, Mark Donowitz, Mary K. Estes Duration: 14 days 13 phases Cell source: Human Primary Intestinal Epithelial Cells (Duodenal, Jejunal, and Ileal Biopsies)

Abstract

This protocol describes the establishment and maintenance of human intestinal enteroids (HIEs) from primary intestinal biopsies for the study of human rotavirus infection and pathophysiology. HIEs are three-dimensional, nontransformed cultures containing multiple epithelial cell types (enterocytes, goblet cells, enteroendocrine cells, and Paneth cells) that recapitulate the cellular complexity and physiological properties of the human small intestinal epithelium.

Cell source
Human Primary Intestinal Epithelial Cells (Duodenal, Jejunal, and Ileal Biopsies)
Application
Disease modeling and viral infection studies

Protocol overview

62 steps across 13 phases

Phase 1 · Human Intestinal Tissue Collection and Crypt Isolation Day 0
  1. a Tissue Collection
  2. b Epithelial Crypt Isolation
Phase 2 · Human Intestinal Enteroid Establishment (Expansion Phase) Days 1-5
  1. a Prepare Complete Medium with Growth Factors (CMGF[+] Medium)
  2. b Resuspend Isolated Crypts in Matrigel and CMGF(+) Medium
  3. c Culture Enteroids in CMGF(+) Medium for 4-5 Days
  4. d Passage Enteroids (Optional)
Phase 3 · Human Intestinal Enteroid Differentiation Days 6-10
  1. a Prepare Differentiation Medium
  2. b Replace CMGF(+) Medium with Differentiation Medium
  3. c Verify Differentiation by Morphology and Gene Expression
Phase 4 · Rotavirus Preparation and Trypsin Activation Days 0-2 (Prior to Infection)
  1. a Propagate Rotavirus Strains in MA104 Cells
  2. b Purify Viral Particles (Optional)
  3. c Determine Viral Titer
  4. d Activate Virus with Trypsin
Phase 5 · Rotavirus Infection of Human Intestinal Enteroids Days 11-12
  1. a Prepare Differentiated Enteroids for Infection
  2. b Inoculate Enteroids with Trypsin-Treated Virus
  3. c Viral Adsorption Period
  4. d Remove Virus Inoculum
  5. e Resuspend in Differentiation Medium with Pancreatin
Phase 6 · Viral Infectivity Assessment and Sample Processing Days 12-14
  1. a Harvest Infected Enteroid Samples
  2. b Prepare Samples for Viral Titer Assay
  3. c Quantify Infectious Virus by Fluorescent-Focus Assay (FFA)
  4. d Calculate Fold-Change in Viral Growth
Phase 7 · Assessment of Infected Cell Percentage by Flow Cytometry Days 12-14
  1. a Dissociate Infected Enteroids into Single-Cell Suspension
  2. b Pellet and Resuspend Cells
  3. c Fixation of Cells
  4. d Permeabilization and Antibody Staining
  5. e Secondary Antibody Staining
  6. f Flow Cytometry Analysis
Phase 8 · Enteroid Swelling Assay (Fluid Secretion Assessment) Days 11-12
  1. a Prepare Enteroids in Optical Imaging Plates
  2. b Image Baseline Enteroid Morphology
  3. c Add Test Compound (Virus or Agonist)
  4. d Time-Lapse Imaging
  5. e Image Analysis and Quantification
  6. f Statistical Analysis
Phase 9 · Immunofluorescence Analysis of Infected Cell Types Days 12-14
  1. a Fix Infected Enteroids
  2. b Dehydration and Paraffin Embedding
  3. c Section Preparation
  4. d Antigen Retrieval
  5. e Primary Antibody Staining
  6. f Wash and Secondary Antibody Staining
  7. g Mounting and Confocal Microscopy
  8. h Image Processing and Quantification
Phase 10 · Quantitative Reverse Transcription-PCR Analysis of Cell Type Markers Days 11-12
  1. a Extract Total RNA from Enteroids
  2. b Quantify RNA Quality and Quantity
  3. c Prepare qRT-PCR Reaction Mix
  4. d Perform qRT-PCR
  5. e Analyze Expression Data
Phase 11 · Electron Microscopy Analysis of Infected Enteroids Days 12-14
  1. a Fix Samples for Electron Microscopy
  2. b Quench Fixation
  3. c Post-Fix and Heavy Metal Staining
  4. d Dehydration and Resin Infiltration
  5. e Polymerization
  6. f Ultramicrotomy and Section Preparation
  7. g Post-Staining with Heavy Metals
  8. h Transmission Electron Microscopy Imaging
Phase 12 · FUT2 Genotyping of Intestinal Enteroid Sources Days 0-2 (Prior to Experiments)
  1. a Extract DNA from Enteroids
  2. b Amplify FUT2 Gene Fragment
  3. c Sequence PCR Product
  4. d Genotype Determination
Phase 13 · Cytotoxicity Assessment Days 12-14
  1. a Assess Lactate Dehydrogenase (LDH) Release
  2. b Measure LDH Activity
  3. c Calculate Percent Cytotoxicity

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Attribution

This SOP was written by Organthis based on the published method in Saxena et al., 2016. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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