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INTESTINAL Publication-derived

Gastric Models for Infection Studies

Full SOP with Academic or a plan

Source Bartfeld et al. · Max Planck Institute for Infection Biology, Berlin, Germany · 10.1053/j.gastro.2014.09.042

Authors: Bartfeld S, Bayram T, van de Wetering M, Huch M, Begthel H, Kujala P, Vries R, Peters PJ, Clevers H Duration: 14 days 7 phases Cell source: Primary (Human Gastric Biopsy-Derived Organoids)

Abstract

Established the first human organoid model of H. pylori infection demonstrating active CagA oncoprotein injection via the Type IV secretion system into gastric epithelial cells. Infected organoids showed characteristic cell elongation and NF-κB inflammatory signaling, recapitulating key pathogenic events.

Cell source
Primary (Human Gastric Biopsy-Derived Organoids)
Application
H. pylori Pathogenesis

Protocol overview

55 steps across 7 phases

Phase 1 · Tissue Collection and Gland Isolation Day 0
  1. a Obtain human gastric corpus tissue
  2. b Extract glands using EDTA chelation
  3. c Enzymatic digestion of cancer tissue (if applicable)
  4. d Wash digested cells
Phase 2 · Organoid Culture Establishment and Maintenance Days 0-7
  1. a Seed glands or cells in Matrigel
  2. b Prepare complete culture medium (ENRWFG_Ti)
  3. c Add RHOK inhibitor post-seeding
  4. d Culture organoids at 37°C in 5% CO₂
  5. e Monitor organoid formation
  6. f Perform first medium change
  7. g Continue medium changes every 3-4 days
Phase 3 · Organoid Expansion and Serial Passage Weeks 2-4+
  1. a Prepare organoids for passage (Day 7-10 of culture)
  2. b Remove culture medium
  3. c Wash with cold phosphate-buffered saline (PBS)
  4. d Mechanically and enzymatically dissociate organoids
  5. e Collect dissociated organoid suspension
  6. f Centrifuge and wash
  7. g Count viable cells
  8. h Seed dissociated cells at 1:5 split ratio
  9. i Overlay with fresh ENRWFG_Ti medium
  10. j Repeat passages every 1-2 weeks
Phase 4 · Directed Differentiation Protocol (Optional) Days 0-14
  1. a Generate gland-type organoids (Wnt-dependent)
  2. b Induce pit-type differentiation by Wnt withdrawal
  3. c Verify differentiation phenotype
  4. d Generate complete-type organoids
Phase 5 · Bacterial Infection Protocol (H. pylori) Days 0-10 (organoid preparation); Day 10+ (infection)
  1. a Prepare organoids in antibiotic-free medium
  2. b Prepare H. pylori bacterial culture
  3. c Calculate multiplicity of infection (MOI) for microinjection
  4. d Perform microinjection of H. pylori into organoids
  5. e Verify successful injection
  6. f Assess bacterial viability post-injection
  7. g Culture infected organoids
  8. h Harvest infected organoids for downstream analysis
  9. i Prepare controls for infection studies
  10. j Test alternative inflammatory stimuli (optional)
Phase 6 · Single-Cell Isolation and Clonal Organoid Generation Days 0-7 (cell sorting); Days 0-10+ (organoid growth from single cells)
  1. a Dissociate organoids into single cells
  2. b Count cells and assess viability
  3. c Prepare cells for fluorescence-activated cell sorting (FACS)
  4. d Gate on singlets using forward scatter (FSC)
  5. e Sort single cells into culture wells
  6. f Verify sort quality by microscopy
  7. g Seed sorted single cells in Matrigel with RHOKi
  8. h Monitor single-cell organoid formation
  9. i Perform first passage of single-cell-derived organoids
  10. j Characterize clonal organoids
Phase 7 · Tumor Organoid Culture from Gastric Cancer Tissue Days 0-7
  1. a Isolate and prepare cancer tissue
  2. b Enzymatic digestion of cancer tissue
  3. c Wash dissociated cancer cells
  4. d Seed cancer cells in Matrigel
  5. e Overlay with complete culture medium (ENRWFG_A)
  6. f Culture and maintain tumor organoids
  7. g Perform karyotyping on tumor organoids
  8. h Characterize tumor organoid morphology and marker expression
  9. i Functional characterization: p53 pathway assessment (optional)
  10. j Long-term expansion and drug testing

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Attribution

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

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