The overall goal of this procedure is to assess individual CFTR function in an efficacy of CFTR modulating treatments using a forskolin-induced swelling, or FIS assay, in intestinal organoids generated from Cystic Fibrosis patients. This method addresses key questions in the Cystic Fibrosis field. Most notably it helps the identification of patients that can benefit long term from existing or experimental drugs.
The main advantage of this technique is that easily accessible tissue can be used to generate organoids from any CF patient independent of age. The implications of this technique extend to orthotherapy of Cystic Fibrosis, and addresses an urgent current need for testing drug efficacy in a cost effective and individual manner. Demonstrating the procedure will be Marvin Statia from Hubrecht Organoid Technology and Annelot Vonk from Jeffrey Beekman group.
For obtaining optimal forskolin-induced swelling assay data, it is essential to work with a culture containing big organoids with multiple buddings. After identifying such a culture, label one 15 milliliter conical tube with the sample name and one tube as washed. Next, use a p1000 pipette to carefully aspirate the medium from the organoid culture wells without disturbing the basement membrane matrix drops.
And add one milliliter of basal medium to each well. Now use the pipette to break up the basement membrane matrix drops in each well, and transfer the resulting organoid suspensions to the 15 milliliter tube with the sample name. Rinse each well with another millimeter of basal medium and pull the washes in the sample tube.
When all of the samples have been collected, fill the sample tube to a final volume of 12 milliliters with basal medium and use a pre-wet five milliliter pipette to mix the solution. Next, centrafuge the organoids, re-suspending the pellet in one milliliter of fresh basal medium. Cover the same p1000 pipette tip with a p10 tip without a filter and try to rate the suspension 20 times.
Then discard both tips and use a five milliliter pipette to add four milliliters of fresh basal medium to the sample. Tilt the tube about 70 degrees, and vigorously mix the solution two to three times with the new pre-wet p1000 pipette tip. Hold the tube in the tilted position for 10 seconds.
Then use the same p1000 pipette to transfer the top one milliliter of the sample into the washed tubes four times. After the last sample has been harvested, collect the organoids by centrifugation and re-suspend the pellet in 120 microliters of 50%basement membrane matrix. Then place a three microliter droplet onto a glass microscope slide and confirm the presence of at least 30 to 50 organoids within the droplet under a light microscope.
Next, plate three microliter oraganoid droplets onto the middle of each well of a 96 well plate, and place the plate in a 37 degrees Celsius incubator for 15 minutes to solidify the basement membrane matrix. Then add either 100 microliters of full colon medium plus VX809, or a full colon medium alone to the appropriate organoid wells, and return the plate to the incubator for 18 to 24 hours. The next day, use a repeater pipette to add 10 microliters of calcium solution to each well, re-suspending the wells one time with a multi-channel pipette to insure that they are mixing, and return the plate to the incubator for another 30 minutes.
While the cells are being stained, preset the live imaging tool on the live cell imaging confocal microscope to 37 degrees and five percent carbon dioxide to pre-incubate the imaging chamber. At the end of the incubation, transfer the plate to the plate holder on the confocal microscope and use the smart setup option to select the Alexa floor 488 track. Next, set the five ex-lens and scan area to 0.6x to zoom out and capture the entire well.
Adjust the laser power and detector sensitivity to enable the optimal detection of the calcium green labeled organoids over the background. And set the bit depth to eight and the frame size to 512 by 512 pixels. Use the time series option to set the measurement time, frequency, and intervals.
And the tile option to manually determine the individual well positions. Now add 100 microliters of freshly prepared forskolin, and or VX770 to the corresponding wells, starting with the first well to be imaged, and begin the measurement. Due to the dysfunction of the Cystic Fibrosis transmembrane conductance regulator, or CFTR, most of the colon Cystic Fibrosis organoids are compact in size and either do not swell or demonstrate very little swelling 60 minutes after forskolin stimulation, depending on their genotype.
However, when CF organoids are treated with CFTR modulators before forskolin stimulation, the organoids exhibit an increase in swelling. To quantify the swelling, the total calcium green surface areas can be measured at each time point. Once mastered, these techniques can be complete in three or four hours if they are performed properly.
Before attempting this procedure, the most important step is the optimization of the culture conditions for intestinal organoids, since good quality cultures will determine the optimal performance of the FIS assay. After watching this video, you shall have a good understanding of how to work with intestinal organoid cultures and subsequently measure CFTR activity and CFDR modulators response using a forskolin-induced swelling assay.
