In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Summary

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

Abstract

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

Introduction

Intestinal epithelial cells are not only responsible for the absorption of nutrients, but also form an important barrier to defend against pathogenic microorganisms and microbial toxins. This intestinal barrier function is regulated by tight junction permeability and epithelial cell integrity^1,2,3, and dysfunction of the epithelial barrier function is associated with inflammatory bowel disease (IBD). The perijunctional actomyosin ring (PAMR) lies within the cell that is closely contiguous to the tight junctions. The contraction of the PAMR, which is regulated by the myosin li....

Protocol

This study was approved by the Animal Care and Use Protocol of Cambridge-Suda Genomic Resource Center (CAM-SU), Soochow University.

1. Plating and Maintenance of Caco-2bbe on Porous Polycarbonate Membranes

1. Grow cells in a T75 flask with media (DMEM containing 10% FBS). Flasks should be fed regularly, depending on the cell density.
   NOTE: For optimal plating, cells should divide rapidly and have a flat "fried-egg" shape, which indicates that cells are in the growth phase.
2. Once cells are 80% confluent, take the flask out of the incubator and remove the media. Rinse any residual media with 1-2 mL of sterile PBS (witho....

Results

In culture, Caco-2BBe cells grow as a monolayer and slowly differentiate into mature absorptive enterocytes that have brush borders. In this protocol, Caco-2BBe cells were plated with a high density on polycarbonate membranes, and cells reached 100% confluency one day after seeding. However, cells are undifferentiated at this stage: To fully differentiate the cells, the media is changed every 2-3 days for 3 weeks. Cells were stained with nuclei and F-actin stains to show the differences b.......

Discussion

There are several critical steps in the protocol. Caco-2BBe (brush border-expressing) cells are always used for TER measurement, selected from the Caco-2 cell line for expression of brush-border proteins. Caco-2BBe cells have a villus absorptive phenotype when fully-differentiated (after about 3 weeks of culture post-confluence)¹⁷. It is necessary to avoid contamination during the measurement, and to sterilize the electrode. Because the procedure is non-sterile, measurements can only be performed .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
22 G gavage needle	VWR	20068-608
4 kDa FITC-dextran	Sigma	46944
Avertin	Sigma	T48402
Black 96-well plates for fluorescence	Fisher	14-245-197A
C57/B6 mice	Nanjing Biomedical Research Institute of Nanjing University
Caco-2BBe cells	ATCC	CRL-2102
Dextran sulphate sodium	MP Biomedicals	2160110
DMED with high glucose and sodium pyruvate	Hyclone	SH30243.01B
Epithelial (Volt/Ohm) Meter	Millicell-ERS	MERS00002
Ethanol	Sinopharm ChemicalReagent	10009218
Falcon tube (15 mL)	Corning	430791
FBS	Gibco	10437-028
Fluorescence microscope	Olympus	FV1000
Fluorometer	Biotek	Synergy 2
HBSS			138 mM NaCl, 0.3 mM Na2HPO4, 0.4 mM MgSO4, 0.5 mM MgCl2, 5.0 mM KCl, 0.3 mM KH2PO4, 15.0 mM HEPES, 1.3 mM CaCl2, 25 mM glucose
IFNg	PeproTech	315-05-20
Modular Tissue Embedding Center	Leica	EG1150H
Serum collection tubes	Sarstedt	41.1378.005
T75 flask	corning	430641
TNF	PeproTech	315-01A
Parraffin	Sigma	A6330-1CS
Polycarbonate membranes (Transwell)	Costar	3413
Pressure pump	AUTOSCIENCE	AP-9925
Rotary Microtomy	Leica	RM2235
Trypsin-EDTA	Gibco	25200-056
Xylene	Sinopharm ChemicalReagent	10023418