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INTESTINAL Publication-derived

A primary human macrophage-enteroid co-culture model to investigate mucosal gut physiology and host-pathogen interactions

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

👤 Gaelle Noel, Nicholas W. Baetz, Janet F. Staab, Mark Donowitz, Olga Kovbasnjuk, Marcela F. Pasetti, Nicholas C. Zachos ⏱ 21 days 📋 5 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Noel et al., 2017. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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