The overall goal of this procedure is to culture undifferentiated nasal epithelial cells at the air liquid interface. This is accomplished by first obtaining superficial nasal epithelial cells from a human volunteer. The second step is to seed the cells on tissue culture plates and then expand the cells in flasks.
Next, the expanded cells are seeded into transwells and grown to co fluency. The final step is to remove the apical tissue culture medium of the confluent cell cultures on the transwells to establish air liquid interface culture conditions. Ultimately, cells grown at the air liquid interface for three to four weeks will redifferentiate into a mucociliary nasal epithelial cell culture, mimicking the phenotype of the nasal epithelium in vivo.
The main advantage of this technique over existing methods like culturing, bronchial epithelial cells, or obtaining commercially available respiratory epithelial cells is the ability of the investigator to set up priori the population from which to sample the nasal epithelial cells. Nasal biopsies are much less invasive than bronchial brush biopsies during a bronchoscopy and therefore disease populations with moderate to severe disease can be sampled without any significant side effects. While it's important to remember this technique is very useful for studying the effects of inhaled agents on normal respiratory epithelium, it's also important to remember that these samples come each from a unique genetic complement, and this makes them important resources for studying genetic diseases such as cystic fibrosis and primary ciliary dyskinesia.
Demonstrating the procedure will be Missy Brighton, a technician from my lab. In preparation for the nasal scrape biopsy seat, the subject upright in a straight backed chair with head tilted back slightly superficial epithelial cells lining the nasal terminates will be obtained under direct vision through a nine millimeter reusable polypropylene nasal speculum on an operating auto scope with speculum. This device provides optimal visualization of the nasal terminates and flexibility of motion.
Insert the auto scope speculum into the nostril and the inferior terminate visualized with illumination. Insert a sterile thermoplastic curette through the speculum with the tip extended distally to the back of the terminate using gentle pressure on the inferior surface of the terminate. Draw the curette across the mucosal surface five times and then retract the curette.
A successful retrieval of mucosal cells held by capillary action will be evident in the cup of the curette. Place the curette with the cells in a 15 milliliter conical tube containing eight milliliters of RPMI 1 6 4 0, and place on ice for transport to the laboratory. Use forceps to retrieve the curette with the cells and then use a P 1000 pipette to dislodge the cells from the curette.
Discard the curette. The nasal epithelial cells obtained by a nasal scrape biopsy are subsequently seated in a pure cold coded 12 well plate. To begin this procedure at a minimum volume of bronchial epithelial cell growth medium or BEGM plus plus about 80 to 100 microliters per well into one to four wells of the coated plate, depending on biopsy size.
Next, use a P 1000 pipette to carefully remove the pellet of the biopsy tissue from the tube without aspirating too much media. Transfer the pellet into the middle of the wells that contain media. Keep the biopsy together as a cluster of cells.
Do not break it up. To make a single cell suspension. A good biopsy should yield up to four wells that can be seeded.
Put the plate into a tissue culture incubator culture. The cells following the protocol and the accompanying manuscript, The cells seated in the 12 well plate are monitored daily until they reach 80 to 90%co fluency, at which point they're transferred to flasks for expansion. Carefully aspirate media from each well and then add 500 microliters of point 25%trypsin per.Well.
Keep the plate at 37 degrees Celsius until the cells are detached. This usually takes two to three minutes dislodge cells by pipetting up and down, and then transfer detached cells to a 15 milliliter conical tube containing soybean tripsin inhibitor or SBTI centrifuge to pellet after aspirating. The S natin Resus suspend in one milliliter, BEGM plus media and vortex the tube expand the cells in a T 25 or T 75 flask, depending on pellet size.
A T 75 flask is used in this demonstration. Add five milliliters, BEGM plus media to the flask, and then add the cell suspension to the flask. Transfer the flask to the tissue culture incubator.
After expanding NECs in flasks, the next step is to seed cells on tissue culture inserts. Remove the media from the T 75 flask and add four milliliters. Trypsin incubate cells at 37 degrees Celsius for two to three minutes until the cells are detached.
Dislodge cells by tapping the flask and pipetting up and down. Transfer to a 15 milliliter tube containing SBTI. Rinse the flask with two milliliters.
Hank's, balance salt solution and add the to the 15 milliliter tube, centrifuge the pellet, and then remove the supra natin. Carefully prepare the 12 well plates. After deciding how many plates are going to be seeded, label the plates with the same code and number, passage number and date.
Add 700 microliters, BEGM plus media to the basal chamber of each. Well resuspend the cell pellet in one milliliter, B-E-G-M-A-L-I. Media by vortexing the tube.
Count the cells with the hemo cytometer, and I loop the cell suspension with B-E-G-M-A-L-I media to the total volume needed for the amount of plates that should be seeded. Add 200 microliters of cell suspension to the apical side of each uncoated. 12 well cell insert.
Transfer the plates to the tissue culture incubator to maintain the cell cultures. Change the media every other day. Use B-E-G-M-A-L-I media, 700 microliters for the basal compartment, and 200 microliters for the apical compartment.
Inserts have to be maintained submerged until cells are totally confluent. Two to six days after seeding on tissue culture inserts, the cells should be completely confluent with no holes visible in the monolayer to establish air liquid interface or a LI 48 hours after the induction with retinoic acid. Remove all media and add 700 microliters pneumoccal, a LI, maintenance media to the basolateral side only.
There is no medium on the apical side. Maintain the cell cultures for four weeks. At the A LI.Please refer to the accompanying protocol text for further details of the procedure for establishing and maintaining a LI, human NECs cultured following The protocol demonstrated in this video redifferentiate into a heterogeneous layer of ECS composed of ciliated and non-rated cells representing the in vivo situation.
The NECs were fixed in 4%FA and embedded in paraffin NECs. Cultures stained with hematin and eosin are shown in panel A.Some of the differentiated NECs are mucus producing goblet cells identified in blue in panel B by Ian Blue. Periodic acid shift staining this image of a hematin and eosin stain paraffin embedded section of A NEC culture shows examples of suboptimal undifferentiated NECs using a different protocol and media formulations.
In contrast to what was observed in the previous figure, the cells here are squamous and neither cuboidal nor affiliated, and do not mimic a pseudos stratified respiratory epithelium. A key point in all of these procedures is the first one, and that is the acquisition of a really good sample of nasal epithelium. This leads to the development of the whole culture system and the development of good differentiated epithelium in vitro Following this procedure.
Methods like R-T-P-C-R, Western blotting, EIA, as well as numerous other cellular and biochemical techniques can be used to answer questions related to the responses induced by inhaled agents or pathogens. Not every nasal scrape biopsy will yield successful cultures. This will depend on the condition of the subject as well as the condition of the tissue collected.
