read_status: annotated
type: paper
title: Co‐Culture System of Human Enteroids/Colonoids with Innate Immune Cells
year: 2020
authors:
- Staab, Janet F.
- Lemme‐Dumit, Jose M.
- Latanich, Rachel
- Pasetti, Marcella F.
- Zachos, Nicholas C.
journal: Current Protocols in Immunology
doi: 10.1002/cpim.113
url: https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpim.113
project:
- Rotation_03
system:
- intestinal_epithelium
- epithelial_barrier
- basolateral_compartment
- apical_compartment
- innate_immune_niche
disease:
- intestinal_inflammation
- infectious_diarrhea
- mucosal_barrier_dysfunction
genes:
- m_csf
- wnt3a
- r_spondin_1
- noggin
- egf
processes:
- epithelial_immune_crosstalk
- epithelial_barrier_regulation
- innate_immune_activation
- cytokine_secretion
- immune_cell_transmigration
- epithelial_differentiation
methods:
- human_enteroid_monolayers
- transwell_co_culture
- primary_immune_cell_isolation
- teer_measurement
- cytokine_profiling
- immunofluorescence_imaging
key_findings:
- immune_cells_modulate_epithelial_barrier
- compartmentalized_epithelial_immune_signaling
- neutrophil_transmigration_across_epithelium
- macrophage_epithelial_homeostatic_interactions
limitations:
- short_term_co_culture_viability
- absence_of_mesenchymal_and_neuronal_cells
- reduced_systemic_immune_complexity
relevance:
- physiologically_relevant_human_gut_model
- platform_for_host_pathogen_studies
- translational_preclinical_assay
tags:
- gi_models
- organoids
- innate_immunity
- epithelial_barrier
concepts:
- enteroid_monolayer_co_culture
- epithelial_barrier_integrity
- epithelial_immune_crosstalk
- transwell_polarized_assays
Human intestinal enteroids derived from adult stem cells offer a relevant ex vivo system to study biological processes of the human gut. They recreate cellular and functional features of the intestinal epithelium of the small intestine (enteroids) or colon (colonoids) albeit limited by the lack of associated cell types that help maintain tissue homeostasis and respond to external challenges. In the gut, innate immune cells interact with the epithelium, support barrier function, and deploy effector functions. We have established a co-culture system of enteroid/colonoid monolayers and underlying macrophages and polymorphonuclear neutrophils to recapitulate the cellular framework of the human intestinal epithelial niche. Enteroids are generated from biopsies or resected tissue from any segment of the human gut and maintained in long-term cultures as three-dimensional structures through supplementation of stem cell growth factors. Immune cells are isolated from fresh human whole blood or frozen peripheral blood mononuclear cells (PBMC). Monocytes from PBMC are differentiated into macrophages by cytokine stimulation prior to co-culture. The methods are divided into the two main components of the model: (1) generating enteroid/colonoid monolayers and isolating immune cells and (2) assembly of enteroid/ colonoid-immune cell co-cultures with separate apical and basolateral compartments. Co-cultures containing macrophages can be maintained for 48 hr while those involving neutrophils, due to their shorter life span, remain viable for 4 hr. Enteroid-immune co-cultures enable multiple outcome measures, including transepithelial resistance, production of cytokines/chemokines, phenotypic analysis of immune cells, tissue immunofluorescence imaging, protein or mRNA expression, antigen or microbe uptake, and other cellular functions.
Human enteroid/colonoid monolayers can be stably co-cultured with primary macrophages or neutrophils
Basolateral positioning of immune cells preserves epithelial polarity and enables physiologic signaling
Macrophage co-culture supports epithelial barrier integrity and cytokine exchange over 24–48 hr
Neutrophil co-culture models acute inflammatory interactions and immune migration across epithelium
The system enables segment-specific epithelial responses using patient-derived tissues
Generation of adult stem-cell–derived enteroid monolayers on Transwell inserts
PBMC isolation followed by monocyte differentiation into macrophages using M-CSF
Primary neutrophil isolation from human blood for short-term co-culture
TEER measurements to quantify epithelial barrier integrity
Multiparametric outputs: cytokine assays, immune phenotyping, imaging, uptake assays
Establishes a human-relevant epithelial–immune niche absent from epithelial monocultures
Reframes enteroids from epithelial-only systems to modular multicellular platforms
Enables mechanistic dissection of immune contributions to epithelial physiology
Supports personalized medicine approaches using matched epithelial and immune donors
Figure 1: Schematic of polarized enteroid–immune cell Transwell co-culture architecture
Figure 2: TEER dynamics demonstrating epithelial barrier maturation and immune modulation
Figure 3: Cytokine and chemokine output from epithelial–immune interactions
Figure 4: Immunofluorescence localization of immune cells relative to epithelial monolayers
Neutrophil co-cultures restricted to very short experimental windows
Absence of adaptive immune components limits chronic disease modeling
Reductionist architecture lacks mesenchymal and neuronal contributions
Immune-derived cytokines cannot always be unambiguously assigned to specific cell types