人間の腸 Organoids 上皮関門の透過性のリアルタイム計測

The overall goal of this procedure is to measure epithelial barrier permeability in human intestinal organoids over time. This method can help answer key questions in the gastroenterology field, such as how specific toxins, bacterial products or even pharmacologic treatments can alter epithelial barrier permeability. The main advantage of this technique is that it allows for both visualization and quantitative measure epithelial barrier permeability in real time and using human intestinal epithelial tissue.

Prior to microinjecting human intestinal organoids, the microinjector must be set up, and the microcapillary prepared, installed and tested as described in the text protocol. To fill the microcapillary with the injection material, submerge the tip of the glass microcapillary in the FITC-dextran injection suspension, being careful to avoid breaking the tip of the microcapillary against the side or bottom of the tube. Pull back on the mineral oil syringe to draw approximately 10 microliters of the FITC-dextran suspension into the microcapillary.

When the microinjection suspension fills 90%of the length of the glass microcapillary, stop filling the microcapillary. Depress the syringe slightly before withdrawing the microcapillary from the microinjection solution to ensure that the tip of the microcapillary does not contain pockets of air. Remove the human intestinal organoid culture plate from the cell culture incubator and transfer to the bio safety cabinet with the microinjector.

Remove the lid from the culture plate and center the first well on the microscope stage so that it is clearly visible through the eyepiece of the scope at the lowest magnification setting. Turn the micromanipulator arm such that the microcapillary is pointed down into the culture well at an angle greater than 45 degrees relative to the microscope stage. Position the tip of the microcapillary above the first culture well at approximately one centimeter above the surface of the media.

Check that both the tip of the glass filament and the human intestinal organoid are visible through the eyepiece. The key to a successful microinjection is to carefully line up the microcapillary with the organoid before attempting the microinjection. And I always recommend that new users practice with spare tissue before they attempt a critical experiment.

By turning the z axis control knob clockwise, advance the microcapillary slowly. Pierce the human intestinal organoid with the microcapillary tip. The outer surface of the human intestinal organoid will depress slightly as the microcapillary begins to apply pressure, and will pop back into shape as the tip penetrates the lumen.

If there is difficulty seeing the tip of the microcapillary within the lumen, adjust the lighting or magnification settings of the dissecting microscope. When the microcapillary is correctly positioned with the tip of the microcapillary near the center of the human intestinal organoid, remove hands from the micromanipulator controls. Depress the mineral oil filled syringe slightly to push the microinjection solution out of the microcapillary and into the human intestinal organoid lumen.

The human intestinal organoid may expand slightly to accommodate the volume of the injection. With the microcapillary positioned above the media to avoid accidental damage to the human intestinal organoids during maneuvering, move to the next human intestinal organoid target. Between treatments or in the event of breakage, change the microcapillary.

Loosen the clamp on the end of the micromanipulator arm and remove the microcapillary from the micro pipette holder. To test compounds delivered to the apical epithelium, to compound should be re-suspended in sterile PBS containing two milligrams per milliliter FITC-dextran. In this representative experiment, re-suspend Clostridium difficile toxin TcdA, add 12.8 nanograms per microliter in PBS containing FITC-dextran prior to microinjection.

Microinject the compound into the human intestinal organoid lumen as demonstrated in the previous section. To test compounds delivered to the basal lateral compartment, after microinjection of FITC-dextran, replace the external culture media with new media containing two millimolar EGTA as the positive control, PBS vehicle alone as a negative control, or other experimental compound. Begin live imaging immediately following microinjection.

This representative bright field image shows a tissue-derived human intestinal organoid after microinjection of FITC-dextran. The fluorescent signal is apparent even without the use of a specific filter set. Stem cell-derived human intestinal organoids were microinjected with four kilodalton FITC conjugated dextran suspended in PBS, or PBS containing Clostridium difficile toxin TcdA.

As a positive control, EGTA was added to the human intestinal organoid culture media in a subset of human intestinal organoids injected with PBS and FITC-dextran. The human intestinal organoids were imaged for 20 hours. As illustrated by these images, human intestinal organoids injected with PBS retained nearly all the fluorescent signal present at T zero.

However, human intestinal organoids that were also injected with TcdA, or treated with EGTA, exhibited a substantial decrease in fluorescent intensity by eight hours post microinjection. The imaging data was quantified for all human intestinal organoids at all time points to generate a high-resolution data set representing the relative change in fluorescent intensity over time in each experimental condition. Once mastered, this technique can be done in approximately 30 minutes to one hour depending upon the number of experimental conditions.

While attempting this procedure, it's important to remember to be patient and take your time when aligning the human intestinal organoids with the microcapillary prior to microinjection. Following this procedure, other methods like PCR, western blot, and RNA seek can be performed to answer additional questions regarding the response of the human intestinal organoid to the injected material. Development of this technique allowed us to examine the effects of bacterial toxins, pharmacologic agents, cytokines, and even live microorganisms on epithelial barrier function in human intestinal organoids.

After watching this video, you should have a good understanding of how to microinject fluorescently labeled dextran and other experimental materials into the lumen of a human intestinal organoid. Don't forget that when working with bacterial toxins or live organisms, it can be extremely hazardous, and so precautions such as using appropriate PPE should always be taken while performing this procedure. And lastly, be especially careful to avoid puncture wounds as the microcapillaries are extremely sharp.