Measuring Intestinal Barrier Permeability by Detecting Flux of Fluorescent Tracers Across Human Colonoid Monolayers

Summary

We describe a technique for measuring intestinal barrier integrity, whereby the rate of translocation of a fluorescently-tagged sugar of known size reflects the permeability of tissue-derived human intestinal colonoids.

Abstract

The intestinal barrier is a critical site of regulation between luminal antigens and the host immune system, and its dysregulation is implicated in multiple gastrointestinal diseases. Intestinal organoids can be a useful tool to understand and model differences in intestinal barrier integrity. Here, we describe a protocol that uses human colonoids to quantify functional intestinal barrier differences by measuring the flux of fluorescent tracers across organoid monolayers. After growing monolayers to confluence, we add a fluorescent tracer, such as FITC-dextran, to the apical chamber and detect its translocation into the basolateral chamber over time. Increased translocation of the fluorescent tracer is reflective of a more permeable, or weaker, epithelial barrier. This protocol is an adaptable platform that allows evaluation of functional intestinal barrier differences across multiple conditions in parallel. Using variations of this protocol, one can assess transepithelial permeability to molecules of various size, shape, and chemical structure, as well as permeability differences in the context of isolated treatments or microenvironmental changes on the apical or basolateral sides of epithelial cells. This allows for a deeper understanding of the mechanism and treatment of barrier disruption in disease states.

Introduction

The intestinal mucosa is a complex structure that plays an important role in regulating the contact between the metabolites, antigens, and microbes in the intestinal lumen and the immune system and enteric circulation. Intestinal epithelial cells are a key component of this system, and in healthy conditions, form a highly selective barrier. Transport across the epithelium can occur through multiple mechanisms: transcellular active or passive transport, paracellular pore or leak pathways regulated by tight junctions and adherens junctions. Whether any of these pathways can be used by a molecule to translocate across the epithelium is determined by the hydrophobicity, s....

Protocol

All samples were acquired and used in accordance with a protocol approved by the University of Colorado Institutional Review Board (IRB), protocol number 14-2012.

NOTE: All procedures in steps 1-3 should be done in a sterile biosafety cabinet with sterile technique. A schematic of the protocol is presented in Figure 1.

1. Establishment of human colonoids from frozen cultures

NOTE: Ensure all materials are ready in the biosafety cabinet before thawing cells to minimize exposure of thawed organoids to freezing medium. The protocol begins with ....

Results

Using this protocol, the permeability of healthy human colonoids in the presence and absence of inflammatory signaling was analyzed. Addition of a mixture of inflammatory cytokines, termed cytomix, which includes IFNγ, TNFα, and IL-1β (each at 10 ng/ml), has been previously shown to induce intestinal barrier dysfunction as measured by TEER in intestinal epithelial cell models^20,27. We aimed to determine if this barrier dysfunction could be recapitulated using the.......

Discussion

This is a protocol to assess the intestinal barrier in the physiologically representative model of human colonoid monolayers. The fluorescent tracer flux assay described here provides information about the functional integrity of the barrier beyond what is gleaned with other types of barrier assays, such as TEER, Ussing chamber measurements, or evaluation of tight junction protein expression. Additionally, the use of patient-derived intestinal organoid models confers the benefit of recapitulating complex human physiology.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL conical centrifuge tubes	Celltreat	229411
2 mL Internal Threaded Cryogenic Vial	Corning	430488
50 mL conical centrifuge tubes	Celltreat	229421
6-well plate, tissue culture treated	Celltreat	229106
70 μm cell strainer	Falcon	352350
96-well black/clear bottom plate	Thermo Scientific	165305
A83-01	R&D	2939
BioTek Synergy plate reader	Agilent	Synergy H1
Cellometer Auto T4 Brightfield cell counter	Nexcelcom	CMT-AT4P
Cellometer cell counting chambers	Nexcelcom	NC1531583
CHIR99021	Sigma-Aldrich	SML1046-5MG
Collagen coating solution	Cell Applications	125-50
DMEM/F-12	Gibco	11330057
DMSO	Sigma-Aldrich	D2650-100ML
Ethanol 100%	Fisher Scientific	A4094
EVOM2 Voltohm Meter with Stx2 Electrode	World Precision Instruments	NC9792051
Fetal Bovine Serum (FBS)	Cytiva	SH30396.03HI
Filter pipet tips	Fisher Scientific	02-707-000, 02-707-002, 02-707-006, 02-707-008
Fluorescein isothiocyanate (FITC)-dextran, 4kDa	Sigma-Aldrich	46944
Gastrin I (Human)	R&D	3006
GlutaMAX	Fisher Scientific	35050061	200 mM L-alanyl-L-glutamine dipeptide
Hanks buffered saline solution 10x	Thermo Scientific	14065056
HEPES 1M	Thermo Scientific	15630080
Interferon gamma recombinant human	BioLegend	575308
Interleukin 1 beta recombinant human	Thermo scientific	200-01B-500UG
IntestiCult Intestinal Organoid Growth Medium (Human)	STEMCELL Technologies	06010	Commercial intestinal organoid media
Live inverted microscope	Olympus	IX85
Matrigel	Corning	354234	Extracellular matrix (ECM)
Microbiological incubator	Fisher Scientific	151030515
Multiwell plate for suspension culture, 24 well	Greiner Bio-one	662102
Nicotinamide	R&D	4106
Parafilm	Millipore Sigma	HS234526B	Sealing film
Pasteur pipets (borosilicate glass)	Fisher Scientific	13-678-20B
PBS	Gibco	10010-023
Penicillin Streptomycin (10,000 U/mL)	Gibco	15140122
Recombinant moouse epidermal growth factor	R&D	2028-EG
Refrigerated centrifuge	Beckman Coulter	BE-AX15R
SB202190	R&D	1264
Thiazovivin	R&D	3845
ThinCert cell culture insert for 24 well plate, 0.4 μm	Greiner Bio-one	662641
Trypsin-EDTA (0.25%)	Fisher Scientific	25200114
Tumor necrosis factor alpha recombinant human	R&D	10291-TA-100
Y-27632 dihydrochloride	Tocris	TB1254-GMP