The significance of this protocol is to test the gross probability of enteroids on in vitro model of premature epithelium. The main advantage of this technique is to measure the permeability from an enclosed lumen that mimics the gut luminal environment. This protocol can be used to study the factors that induce or attenuate leaky gut disease.
It can also be applied to endothelial or vascular systems to study conditions that can induce vascular leakage. This protocol requires some practice. We recommend practicing each section separately before putting the whole procedure together.
To begin, transfer the intestine segments to a 60 by 15 square millimeter Petri dish with ice-cold Dulbecco's PBS with amphoteracin and soak for 20 minutes on ice. Using a scalpel with a pair of scissors, cut the intestine segments into three to five millimeter pieces and place one to two pieces in a 35 by 15 square millimeter Petri dish containing 0.5 to one milliliter of organoid growth medium on ice. Cut the intestinal tube longitudinally using dissecting micro-scissors and forceps.
Then use the forceps to scrape the epithelial cells from the fascia. Remove the fascia and swirl the dish to break the cell clumps. Then, using a 20-microliter pipette cut tip, transfer the cells and the media at an increment of five to 10 microliters to a basement membrane matrix mix to make a 285-microliter solution.
Mix the cells by pipetting in a basement membrane matrix on ice using a 200-microliter cut tip. After mixing, place five 50-microliter strips of the cell basement membrane matrix mix in one well of a pre-warmed six-well plate kept on a warm foam brick. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 10 minutes to allow polymerization and a hardening of the basement membrane matrix.
Once the matrix strips are solidified, add two milliliters of the enteroid growth media into the well before putting the plate back in the incubator. After transferring the enteroids to a well of a six-well or 12-well plate, every alternate day, replace the old media with fresh media. And as the enteroids have started to thrive, pass the cells every five to seven days.
For immunofluorescence staining, add 500 microliters of the primary antibody in blocking buffer to each well of enteroids and incubate overnight at four degrees Celsius. The next day, remove the antibody solution and wash the enteroids thrice with PBS. After incubation in one to 400 diluted secondary antibody solution, followed by incubation in DAPI, wash the enteroids three times with PBS.
To preserve the three-dimensional structures of the enteroids, use cutouts of thin silicon rubber sheets or glass cover slips to create a 0.5 to one millimeter space between the bottom glass slide and the cover slip. For mounting, wash the stained enteroids with 70%glycerol. Then, using a one-microliter inoculating loop, or a cut 200-microliter tip, lift the enteroids out of the plate and mount with glycerol onto a glass microscope slide.
Prepare a Petri dish covered with a transparent film cover and add two to four one-microliter drops of the Dextran-FITC on the film. Using the micro manipulator, drive the micro pipette tip inside the liquid and above the film under a stereo microscope, then gently pull the syringe to withdraw the Dextran-FITC into the micro pipette. After filling the micro pipette with two to four microliters of Dextran-FITC, push the syringe to remove any air from the pipette tip.
Also, inspect the injected material column in the micro pipette to ensure no air pocket and record the volume of Dextran-FITC inside the micro pipette. Next, turn on the air source connected to the pneumatic pump before turning on the pump and setting the pump duration to 10 to 15 milliseconds. Then turn the stopcock on the syringe to open the line from the pump to the micro pipette.
Remove the enteroids from the incubator and place the dish on a warm foam brick in a covered container to minimize light exposure after micro-injection. Place the Petri dish with enteroids under the stereo microscope and move the micro manipulator knobs to place the micro pipette tip at a 35-to 45-degree angle to the horizontal surface. Visualize and identify the spherical enteroids with a target of injecting three to five enteroids per dish.
Next, advance the tip to a target enteroid by looking through the microscope eye pieces. Then, advancing the Z-axis knob with a precise slow movement, puncture the enteroid with the micro pipette tip. The advancement should stop once the tip goes through the enteroid surface, which depresses and pops back.
Tap on the pump pedal with one foot and fill the enteroid with the greenish Dextran-FITC solution until expanded. Record the number of pumps to calculate the pumped volume and Dextran concentration. This protocol showed the characterization of fetal tissue-derived enteroids by staining various biomarkers specific to intestinal epithelium with immunofluorescent antibodies, confirming their small intestine epithelial origin.
Enteroids at different stages are shown here. A seven-to 10-day-old enteroid is small and thick. Whereas an enteroid ready for micro-injection was large with a lumen and thin wall.
The enteroid two days post-micro-injection still contains significant amounts of Dextran-FITC. The permeability of the enteroids after micro-injection was assessed by measuring the Dextran concentration in the media. EGTA, known to increase membrane permeability at tight junctions, was used as a positive control.
The Dextran measurement assay further show that LPS induced increased permeability and higher LPS concentration induced higher permeability. The most important thing about fixing and staining is not to disrupt the shape of the enteroids or to lose them during the process. Following micro-injection, media can be collected for cytokine assay and enteroids can be harvested for RNA sequencing.
