With the aim of investigating intestinal barrier integrity, we developed a method to measure the intestinal barrier integrity in mouse small intestinal organoids. In contrast, we refer to used monolayer cell culture, our assay is based on three dimensional small intestinal organoids. Adapting a two dimensional assay to our model was essential for its practicality.
The assay is based on organoids cultured in vitro and its application will help to define inducers and inhibitors of the intestinal barrier integrity. The regulation of tight junction proteins is an important feature of all epithelial cells. Our assay enables the function and analysis of epithelial barrier integrity.
To measure the barrier integrity of organoids isolated from mouse intestinal tissue, first precoat all of the centrifugation tubes that will be used for storing the organoids during the plating process with enough 0.1%BSA in PBS to cover all of the plastic surfaces. Immediately remove the BSA solution and place the tubes on ice. Next, thaw the cell matrix solution and organoid culture medium on ice and carefully remove the culture medium from each well of a 48 well organoid culture plate.
Wash each well with one milliliter of cold PBS before vigorously pipetting to dissolve the cell matrices. When relatively homogeneous cell suspensions have been obtained, wash the organoids with fresh PBS two times by centrifugation and resuspend each organoid culture in 40 microliters of cold medium. Dissociate the large organoid structures through a 10 microliter pipette tip five times to collect structures with a 40 to 60 micrometer size and mix each organoid suspension with 40 microliters of freshly prepared cell matrix solution.
Add each organoid cell matrix solution suspension into the center of individual wells of an eight well chambered coverslip and place the slide on an icepack for five minutes. At the end of the incubation, place the slide at 37 degrees Celsius and 5%carbon dioxide for 20 minutes to enable polymerization of the organoid cell matrix structure before adding 150 microliters of organoid culture medium to each well for a 24 hour incubation in the cell culture incubator. At the end of the incubation, treat the positive control wells with 10 nanograms per milliliter of recombinant murine interferon gamma for 48 hours.
To perform a permeability assay, transfer the chambered coverslip to the 37 degree Celsius warmed incubation chamber of an inverted confocal microscope and set the carbon dioxide of the chamber to 5%Lock the coverslip tightly onto the microscope stage and adjust the imaging settings of the microscope to visualize the organoids in one well of the chamber. Then, add three microliters of freshly prepared 100 millimolar Lucifer yellow in 150 microliters of medium to one well for one hour incubation in the microscope chamber. At the end of the incubation, adjust the focus to visualize the lumen of the reference organoid and define the required laser energy for Lucifer yellow excitation and the respective detection sensitivity of the instrument to image the Lucifer yellow signal at 30 to 40%of the available dynamic range of the instrument.
Alternatively, the signal intensity can be modified by changing the exposure time. To find the positions of about 10 organoids with a spherical structure close to the coverslip surface per well by differential interference contrast live imaging, capturing organoids with comparable diameters and focusing on the central slice of the organoids to image the lumens. Record the differential interference contrast and the Lucifer yellow fluorescence of every position to document the shape and auto-fluorescence of each organoid.
When all of the organoids have been imaged, add 150 microliters of medium, including Lucifer yellow, to the Lucifer yellow treatment wells and image all of the wells every five minutes for 70 minutes. At the end of the imaging session, add four microliters of freshly prepared EGTA to 100 microliters of medium. Add the diluted EGTA to the appropriate wells.
Then, record the fluorescence of the organoids in each well every five minutes for 30 minutes. Be sure to practice the handling and culture or organoids in advance as cellular viability and integrity are a prerequisite for the success of the assay. In this representative experiment, after 70 minutes of treatment with Lucifer yellow intraluminal Lucifer yellow fluorescence was only visible in organoids from wild type animals treated with interferon gamma.
Neither unstimulated controls, nor organoids derived from interferon gamma receptor-2 knockout animals showed intraluminal Lucifer yellow fluorescence at the end of the treatment period. The addition of EGTA caused an unspecific breakdown of the intestinal barrier integrity by sequestering tight junction co-factors, resulting in Lucifer yellow take-up and expression in all organoids regardless of origin or treatment conditions. The relative intensity values for the Lucifer yellow fluorescence level within the organoid lumen and outside of each organoid can also be quantified.
Be sure to select organoids with a comparable size around configuration and to select the set axis so that you can imagine the central lumen of the organoid. In addition to using substances inducing intestinal barrier breakdown, we can also investigate strategies for the inhibition of intestinal barrier destruction. As this methodology is based on primary cells from a single animal, it can be used for many assays, reducing the number of animals required for each experiment.
