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INTESTINAL Publication-derived

Intestinal Organoids for Infection Studies (2016)

Full SOP with Academic or a plan

Source Bartfeld, 2016 · Research Centre for Infectious Diseases, Institute of Molecular Infection Biology, University of Würzburg · 10.1016/j.ydbio.2016.09.014

Authors: Sina Bartfeld Duration: 28 days 9 phases Cell source: Human ASC (Adult Stem Cells) and Human iPSC (induced pluripotent stem cells)

Abstract

This protocol describes the culture and infection of gastrointestinal organoids derived from human adult stem cells and pluripotent stem cells to model infectious diseases, inflammatory bowel disease, and host-pathogen interactions. Organoids represent a 3D primary culture system that bridges the gap between 2D monotypic cell lines and animal models, enabling the study of pathogen infection, microbiota interactions, and epithelial responses in a tissue-mimetic environment.

Cell source
Human ASC (Adult Stem Cells) and Human iPSC (induced pluripotent stem cells)
Application
Disease modeling and host-microbe interaction studies

Protocol overview

33 steps across 9 phases

Phase 1 · Organoid Derivation and Initial Culture Setup Day 0-7
  1. a Isolation of Intestinal Crypts
  2. b Prepare Extracellular Matrix (ECM) - Matrigel or Mouse Sarcoma-Derived ECM
  3. c Seed Crypts into ECM
  4. d Add Organoid Culture Medium
Phase 2 · Organoid Expansion and Maintenance Day 7-28 (ongoing)
  1. a Incubate Organoids
  2. b Medium Change
  3. c Passage/Split Organoids
  4. d Assess Organoid Morphology and Cell Differentiation
Phase 3 · Preparation for Infection Studies Day 25-28 (prior to infection)
  1. a Select Organoid Lineage or Differentiation State
  2. b Optional: Transition to Hypoxic Conditions
  3. c Prepare Bacterial, Viral, or Parasitic Inocula
Phase 4 · Bacterial Infection via Microinjection Day 28 onwards (infection day)
  1. a Perform Microinjection of Bacteria
  2. b Incubate Infected Organoids
  3. c Assess Bacterial Viability and Replication
Phase 5 · Viral Infection Day 28 onwards (infection day)
  1. a Prepare Viral Inoculum
  2. b Infect Organoids via Mechanical Disruption to 2D Cultures or Intraluminal Microinjection
  3. c Monitor Viral Replication
Phase 6 · Parasitic Infection Day 28 onwards (infection day)
  1. a Prepare Parasite Inoculum
  2. b Infect Organoids with Parasites
  3. c Assess Parasitic Infection and Morphology
Phase 7 · Assessment of Host Immune and Epithelial Responses Day 28 onwards (during/after infection)
  1. a Harvest Organoids at Defined Timepoints
  2. b Analyze Gene Expression via qPCR or RNA-Seq
  3. c Assess Barrier Function
  4. d Immunofluorescence Analysis
  5. e Measure Antimicrobial Secretion (e.g., Paneth Cell Degranulation)
  6. f Optional: Assess Metabolic Responses and Hypoxia Signaling
Phase 8 · Alternative: 2D Culture System from Organoids Day 25-28 (preparation); Day 28+ (infection)
  1. a Dissociate Organoids into Single Cells
  2. b Seed Organoid-Derived Cells as 2D Monolayers
  3. c Perform 2D Infection with Simplified Addition to Medium
  4. d Detect Viral or Bacterial Infection in 2D Cultures
Phase 9 · Strain-Specific and Patient-Specific Studies Day 28+ (following organoid establishment from biobank)
  1. a Optional: Use Patient-Derived Organoid Biobanks
  2. b Characterize Genetic and Phenotypic Variation
  3. c Perform Drug Screening or Therapeutic Testing

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Attribution

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

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