人鼻上皮细胞球体的生成与个体化囊性纤维膜电导调节的研究

The overall of this procedure is to generate human nasal cell spheroids which can ultimately be used as an assay for functional CFTR studies. This method can help answer key questions in the cystic fibrosis field such as assessing an individual subject's response to CFTR altering drugs in an ex vivo model system. The main advantage of this technique is that a large number of spheroids are generated in a relatively fast timeframe from a small nasal cell sample allowing for extensive testing.

Demonstrating the procedure will be Lauren Strecker and Jessica Moncivaiz, technicians from my laboratory. After processing and expanding Human Nasal Epithelial or HNE cells on feeder fibroblasts according to the text protocol, bring previously prepared expansion medium, differentiation medium, 0.1%Trypsin EDTA solution, and sterile PBS to room temperature over 30 minutes. Then use 70%ethanol to clean the containers and transfer them to a clean biosafety cabinet.

In the biosafety cabinet, use a Pasteur pipette attached to a vacuum line to aspirate and discard the medium from HNE culture. Then with a clean serological pipette, use five milliliters of PBS to wash the dish by adding, swirling, and aspirating the buffer. Add five milliliters of 0.1%Trypsin EDTA solution to the culture and return it to the incubator at 37 degrees Celsius and 5%carbon dioxide for five minutes.

Then under an inverted microscope at four to 10X, check that the cells detach from the dish, become round and float. Gently tap the side of the culture dish to dislodge the cells and if necessary, re-incubate the dish for an additional five minutes until most cells are detached. Next, using a 10 milliliter serological pipette, add five milliliters of expansion medium to the dish and collect all the liquid into a labeled sterile 50 milliliter conical tube.

If cells remain adherent to the culture dish after three rounds of trypsinization, use a sterile cell scraper to gently release the remaining cells and with five milliliters of expansion medium, wash the scraper and dish and collect the contents into the same labeled conical tube. Centrifuge the tube at 360 times g in four degrees Celsius for five minutes. Aspirate the supernatant and use one milliliter of expansion medium to resuspend the cell pellet.

Then use a hemocytometer to count the cells. Thaw the basement membrane matrix on ice per the manufacturer's instructions. Separate an appropriate number of differentially trypsinized passage cells for a final concentration of 500, 000 cells per milliliter of the matrix.

For a four-well plate use 200, 000 cells and collect them into a 1.5 milliliter tube. Next, centrifuge the cell mixture at no more than 360 times g for five minutes. Then using a one milliliter pipette, completely and carefully aspirate and discard the medium, taking care not to disturb the cell pellet.

Keep the tube of cells on ice when not actively handling it. Use a one milliliter pipette tip to add 100 microliters of the basement membrane matrix per planned well to the cells. While not completely emptying the pipette tip to avoid the introduction of air bubbles, pipette up and down to carefully and thoroughly resuspend the cells.

Move quickly to avoid solidification of the matrix. To seed 100 milliliter matrix aliquots into each well of a four-well IVF culture plate, use a clean pair of scissors to trim off the distal three to four millimeters from a 200 microliter pipette tip. Carefully and slowly pipette a 100 microliter drop of cell matrix mixture into the center of each well, ensuring that the drop remains spherical and does not touch the sides of the well.

Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 30 minutes until solid. Then carefully pipette 500 microliters of differentiation medium into each well, taking care not to disturb the matrix drop. Ensure that the medium just covers the drop and add more if necessary.

To culture HNE spheroids, using a one milliliter pipette tip, gently aspirate all medium from the well while avoiding the matrix which will destroy spheroids. Then with a fresh one milliliter pipette tip and while avoiding touching or disturbing the matrix, gently add 500 microliters of differentiation medium to the well around the matrix. Continue to incubate the spheroids, replacing the differentiation medium three times weekly as just demonstrated.

Under an inverted microscope at 20X magnification, evaluate spheroid morphology daily. Spheroids should form within three to five days of plating and reach maturity within approximately 10 days. HNEs should attach to the culture dish and form small islands within 72 hours of seeding.

Examples of good and poor island formation at one week are shown here. Islands should expand to cover the dish over the course of 15 to 30 days. Within the first three to four days of culture, small cystic structures should begin to form in the matrix and mature over approximately 10 days.

If plated at the described density, successful cultures will contain 50 to 100 spheroids per matrix drop. The lumens may be relatively clear as with wild-type CFTR or filled with cellular debris and mucus as in CF spheroids. Masking to delineate the luminal area of the spheroids is demonstrated in these two panels.

Shown here are examples of poorly formed unsuccessful spheroid cultures. As illustrated in these graphs from functional assays, spheroids from subjects with wild-type CFTR should swell over an hour when stimulated and should swell less or shrink if stimulated in the presence of the CFTR inhibitor inh-172. Spheroids from a subject homozygous for F508del-CFTR should either shrink or swell very slightly with increased swelling or less shrinking when pharmacologically corrected with the CFTR modulators VX809 and VX770.

Once mastered, seeding nasal cell spheroids can be done in 15 to 20 minutes if it's performed properly. While attempting this procedure, it's important to remember to maintain clean operating procedures within the biosafety cabinet to minimize the sample contamination risk and to avoid any risks to your own health. Following this procedure, other methods like fixation, staining, and microscopy can be performed in order to answer additional questions about cell morphology and composition.

After watching this video, you should have a good understanding of how to generate primary human nasal cell spheroids which can be utilized as a personalized assay of CFTR function. Don't forget that working with primary human tissues can be hazardous and precautions such as proper personal protective equipment use should always be taken when performing this procedure.