Individualized therapy for cystic fibrosis patients can be achieved by an in vitro disease model to understand the CFTR activity at baseline. Human nasal epithelial organoids, or HNE organoids, produce lumens of varying sizes that correlate with the CFTR activity, distinguishing between CF and non-CF organoids. Here, we have described a methodology for culturing and imaging these HNE organoids in detail.
Dysfunctional epithelial ion transport, particularly that of chloride and bicarbonate, results in a decreased volume of the epithelial lining fluids. This also affects the mucus secretions leading to mucostasis and obstruction. Therefore, our HNE model has been developed for various applications, depending on the investigator's experimental design and resources.
Apart from assessing CFTR activity at baseline or in response to therapeutics, this technique can also be applied to other diseases involving epithelial cell function, especially epithelial cell fluid transport. These methods were developed primarily for use in cystic fibrosis studies. Other studies evaluating the airway epithelia, such as primary ciliary dyskinesia, may also find these methods helpful.
These techniques require attention to detail and precision. They also require careful observation to ensure that the initial expansion of cells is appropriate. Monitor all cultures every day, and the success will be more likely.
To begin, dissociate the nasal brush biopsy into 8 milliters of RPMI media in a 15 milliliter conical tube by passing the cytology brush several times through a one milliliter large-bore pipette tip. Centrifuge the cells at 500 times g for five minutes at four degrees Celsius. Discard the supernatant and then re-suspend the cell pellet in three milliliters of cell detachment solution.
Incubate at room temperature for 16 minutes to digest. Use five milliliters of the expansion media to wash the cells twice. Then, add them to a T75 flask, pre-seeded with irradiated, inactivated, and 50 to 60%confluent 3T3 fibroblasts.
Allow the cells to co-culture and expand in the expansion media for 7 to 14 days. If antibiotics were introduced for cells derived from patients with cystic fibrosis, the media should be replaced with antibiotic-free expansion media after the initial three days of culture, and can continue changing media every two days until 80%confluent. Wash the cells with 1x DPBS.
Then, add 1.5 milliliters of 0.05%trypsin-EDTA into the T75 flask and incubate for four minutes at 37 degrees Celsius to remove the irradiated and inactivated 3T3 fibroblast from the culture. Rinse the T75 flask with 1x DPBS twice to thoroughly wash away any remaining 3T3 fibroblasts. Add 1.5 milliliters of 0.05%trypsin-EDTA into the T75 flask and incubate for 10 minutes at 37 degrees Celsius to detach HNEs.
Neutralize the trypsin with soybean trypsin inhibitor at a one-to-one ratio. Centrifuge the cells at 500 times gi for five minutes. Then, discard the supernatant and wash the cells with five milliliters of expansion media once.
The cells are now ready for seeding to grow organoids. Thaw the organoid culture extracellular matrix or ECM overnight at four degrees Celsius. Cool the 15-well angiogenesis slides overnight at the same temperature.
Next, cool the pipette tips to four degrees Celsius. Coat the slides with five microliters of cold 100%ECM on ice. Place them into a cell culture incubator at 37 degrees Celsius for at least 30 minutes for consolidation.
Count the HNEs harvested from co-culture using a hemocytometer. Then, dilute the cells to 500 cells per microliters in total number, with 20%ECM diluted by differentiation media on ice. Take the ECM coated slides out of the incubator and seed five microliters of the cold cell ECM mixture into each of the wells.
Transfer the slides immediately into a culture incubator at 37 degrees Celsius for an hour to consolidate the cell ECM mixture. After that, take the slides out of the incubator and feed the cells into each well of the 15-well angiogenesis slides with 50 microliters of the differentiation media. Return the slide into the culture incubator at 37 degrees Celsius, changing the media every other day until the organoids are ready for further experiments.
Pre-treat the 8-well glass-bottom chamber slides with cell adhesive for 30 minutes. Discard the solution and air-dry the wells for another 30 minutes. Next, discard the media from the top of the ECM and then add 50 microliters of the cold 1x PBS into each well of the 15-well slides on ice.
Pipette up and down three to five times using 200 microliters of the large-bore pipette tip. Dispense the solution onto the center of a well of the 8-well chamber slides. Remove the excess liquid immediately from the wells by a fine-tip pipette.
Then, place the chamber slide into a 37 degree Celsius incubator for 40 minutes to enhance the organoid adhering to the glass bottom. After 40 minutes, gently wash the organoids with 1x PBS twice and fix them with 300 microliters of 4%paraformaldehyde in each well for 30 minutes set room temperature. Wash twice with 1x PBS and store the organoids in 1x PBS set four degrees Celsius for immunostaining for up to two weeks.
To harvest the organoids for histological studies, remove the media from the culture and add 50 microliters of cold one 1x PBS into each well of the slides on ice. Pipette up and down three to five times using a 200 microliter large-bore pipette tip. Combine all the solutions from the 15-well slide or culture inserts into a 15 milliliter conical tube on ice.
Adjust the total solution volume in the tube to 10 milliliters by adding cold 1x PBS. Centrifuge the tube at four degrees Celsius, 300 times g for five minutes. Then, aspirate out the supernatant and add 60 microliters of warm histogel to mix with the organoid pellet using a 200 microliter large-bore pipette tip.
Transfer the suspension immediately to a histology mold. After consolidating the histogel at room temperature, put the mold block into 4%paraformaldehyde for fixation overnight at four degrees Celsius. Open the software and right click at the bottom of the screen.
Then, select Analysis Controls, followed by Automated Measurement Results. Open the organoid image and select 5 to 10 organoids with visible lumens. Hold right click on the image to open the menu and select Polygonal ROI to outline the full organoid to obtain the organoid's total surface area.
Then, outline the lumen to obtain the lumen area. Repeat this for the remaining organoids in the well, and all the wells in the assay. Finally, export the data to Excel.
Divide the lumen area by the total surface area and average all the organoids from the sample to get the Baseline Lumen Ratio. The bright field images represent HNEs from a successful and unsuccessful samples collection. The HNEs grow well in a large cluster.
In contrast, the HNEs grow poorly in two small clusters surrounding the irradiated 3T3 cells. Organoids in the 15-well slide have more precise and sharper images than those in the culture insert. Besides this, no significant morphological difference was observed in these two culture methods.
Non-CF organoids typically have a larger lumen and more fluid than CF organoids. The HNE staining in organoids from a non-CF, F508del/F508del subject, and the immunofluorescent staining of cilia in an organoid are shown in these images. The cilia stained with acetylated-tubulin, FITC labeled secondary antibody, and the nuclei labeled with DAPI are shown here.
Maximum projection images of the two representative organoids by whole-mount immunofluorescent staining and their corresponding three-dimensional reconstruction images are shown here. Mucus and cilia within the lumen of the organoids are shown. The graphical images represent the forskolin-induced swelling assay to test CFTR function on the primary nasal epithelial cells.
A representative forskolin dose-response experiment of non-CF volunteers is shown here. FSK dose-response is compared with average fractional change at one hour, versus at eight hours, which suggests that the eight hours assay can produce a more significant swelling difference among different FSK doses than those at one hour. The area under the curve can show a minor difference in swelling compared to average fractional change at eight hours.
While attempting this procedure, keep in mind that organoids will be lost during the collection, fixation and staining process. So, meticulous care must be taken during each step and sufficient starting numbers must be obtained to ensure success. Growing organoids in culture inserts can help in this regard, as these techniques are developed.
Here, we have used commercially available reagents and supplies to facilitate expansion to other investigators. A functional assay was also developed that used common microscope techniques and more specialized equipment.
