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INTESTINAL Publication-derived

Small Intestinal 2D Cell Cultures for Infection Studies

Full SOP with Academic or a plan

Source Noel et al., 2017 · Johns Hopkins University School of Medicine; University of Maryland School of Medicine · 10.1038/srep45270

Authors: Gaelle Noel, Nicholas W. Baetz, Janet F. Staab, Mark Donowitz, Olga Kovbasnjuk, Marcela F. Pasetti, Nicholas C. Zachos Duration: 21 days 5 phases Cell source: Human iPSC-derived enteroids; Human monocyte-derived macrophages

Abstract

This protocol establishes a primary human macrophage-enteroid co-culture system combining intestinal epithelial monolayers derived from stem cell-containing crypts with monocyte-derived macrophages to investigate mucosal gut physiology, barrier function, and immune responses to enteric bacterial pathogens (ETEC and EPEC). The model recapitulates key features of human intestinal epithelium in vitro, including cell diversity, barrier integrity, polarized cytokine secretion, and coordinated responses to infection.

Cell source
Human iPSC-derived enteroids; Human monocyte-derived macrophages
Application
Disease modeling; Host-pathogen interaction studies

Protocol overview

29 steps across 5 phases

Phase 1 · Monocyte isolation and macrophage differentiation Day 0-6
  1. a Collect peripheral blood from healthy volunteers
  2. b Isolate peripheral blood mononuclear cells (PBMC)
  3. c Enrich monocytes from PBMC
  4. d Seed monocytes for macrophage differentiation
  5. e Add additional M-CSF on Day 2
  6. f Change complete media on Day 4
  7. g Complete macrophage differentiation
Phase 2 · Human enteroid generation and monolayer establishment Day 0-21
  1. a Obtain intestinal tissue biopsies
  2. b Generate enteroid cysts from isolated crypts
  3. c Pool and triturate enteroid cysts
  4. d Seed enteroid fragments onto permeable inserts
  5. e Culture enteroid monolayers to confluency
  6. f Induce monolayer differentiation
Phase 3 · Establishment of macrophage-enteroid co-cultures Day 0-1 (co-culture setup)
  1. a Prepare enteroid monolayers for co-culture
  2. b Invert enteroid monolayer inserts
  3. c Seed macrophages on the basolateral side
  4. d Return inserts to normal orientation
  5. e Add media to complete co-culture setup
Phase 4 · Bacterial infection of macrophage-enteroid co-cultures Day 0-1 (infection)
  1. a Prepare bacterial inocula
  2. b Adjust bacterial cell density
  3. c Infect co-cultures with bacteria
  4. d Incubate infected co-cultures
  5. e Harvest and analyze infected cultures
Phase 5 · Bacterial killing assay (ETEC susceptibility evaluation) Day 0-1 (assay)
  1. a Infect enteroid monolayers with or without macrophages
  2. b Wash infected monolayers
  3. c Lyse infected cells
  4. d Prepare bacterial serial dilutions
  5. e Plate bacteria and count CFU
  6. f Calculate bacterial killing

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Attribution

This SOP was written by Organthis based on the published method in Noel et al., 2017. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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