The overall goal of this procedure is to grow nasal epithelial cells obtained from children and reprogram them into induced pluripotent stem cells. This method can help answer key questions in the field of aerated disease such as how asthma develops. The main advantage of this technique is that it collects airway epithelial cells from children without stressful procedures.
In culture, these are in a homogeneous population, and serves as an excellent source to generate induced pluripotent stem cells or iPSCs. We first had the idea for this study when we talked to Drs. Ji and Hershey about their study to collect meso-epithelial cells from children to investigate epigenetic variation in asthma and air pollution.
Visual demonstration of these techniques is critical because nasal sampling and reprogramming to iPSCs of nasal epithelial cells are difficult techniques to learn since they're based on personal experience and not established protocols. Prior to the participant's visit, transfer two milliliters of fresh bronchial epithelial growth medium, abbreviated BEGM, to a 15 milliliter conical tube. Then, to this medium, add 20 microliters of a mixture of penicillin, streptomycin, and amphotericin b to a final concentration of 0.01%When the participant arrives, direct them to a chair to sit.
Then open a cytology brush immediately before taking the sample, and do not touch it to any surfaces to ensure that it remains sterile. Proceed to instruct the participant to sit still, and tilt their head upwards towards the ceiling. If they are restless, have them sit on their hands so that they do not attempt to swat the brush away.
Next, aim and insert the brush to the back of the nose where the passage narrows. Afterwards, remove the brush from the nostril by sliding it down with a twisting motion of the wrist. Continue by placing the brush in the previously prepared conical tube, and ensure that it is submerged in BEGM.
Then cut off the excess portion of the brush and fasten the cap on the tube. After the sample has been collected, immediately place it in a beaker filled with warm water so that it is kept as close to 37 degrees Celsius as possible. Then transport it to the laboratory.
Once in the laboratory, gently agitate the brush in the BEGM. Next, remove 10 microliters of the sample and to this, add 10 microliters of a live/dead cell stain. Proceed to introduce this mixture onto a hemocytometer.
And count only the live cells under a microscope. Following cell counting, take 50 microliters of the sample and proceed to dilute it to 200 microliters with 1X PBS. Transfer the remainder of the undiluted cell mixture, along with the brush, to a regulated 37 degree Celsius waterbath until cell seeding.
Proceed to add the PBS-diluted sample to a cytospin funnel attached to a glass slide. And spin the entire aparatus at 400 rpm for two minutes. Afterwards, remove the funnel and allow the slide to dry overnight in the dark.
After staining the slide with Hema 3, as discussed in the text protocol, observe it under a microscope and count the cells. Identify epithelial cells by their unique characteristics. A columnar shape, cilia, and one round nucleus, and determine the percentage of these cells in the preparation.
To begin seeding, gently swirl the brush inside the conical within a certified tissue culture hood, and then remove it from the tube. Afterwards, gently rotate the brush on the inner surface of a T25 flask, previously coated with bovine dermal collagen, taking care not to scratch the coating. Then discard the brush in an appropriate biohazard container.
Slowly pipet the remaining cells in BEGM from the conical into the T25 flask, at least 7 times 10 to the fifth cells. Proceed to observe the cells under an inverted microscope. After culturing and passaging cells, as described in the text, proceed to plate 5 times 10 to the fifth nasal epithelial cells in two milliliters of BEGM per well of a six well tissue culture plate.
Then incubate the cells for 24 hours at 37 degrees Celsius and 5%carbon dioxide. Following incubation, assure that cells are robustly proliferating. Then add Polybrene to each BEGM-containing well to a final concentration of eight micrograms per milliliter under a fume hood.
Proceed to transduce the nasal epithelial cells by adding lentiviral particles, expressing Oct4, Sox2, Klf4, and c-Myc to the medium in each well as discussed in the text protocol. After incubating the transduced cells for approximately three hours, remove the virus-containing medium and discard it, using institutionally approved procedures. Then replace this medium with BEGM, and return the transduced nasal epithelial cells to the incubator.
After three days, replace the medium with fresh BEGM. And then incubate the transduced cells under the same conditions. The next day, add 0.1%gelatin solution to two wells of a six-well plate in order to begin preparing mouse embryonic fibroblast-coated plates.
Then cover the plate with its corresponding lid, and incubate it overnight at 37 degrees Celsius. Proceed on day five, to thaw a vial containing approximately 5 times 10 to the fifth inactivated mouse embryonic fibroblasts, abbreviated as MEFs. Then place these cells into 10 milliliters of MEF medium.
And proceed to spin them at 200 x g for five minutes. Afterwards, aspirate the supernatant and resuspend the MEFs in the same medium. Once these cells have been counted, aspirate the gelatin solution from the previously prepared six-well plate.
Next, add 1.87 times 10 to fifth MEFs into each gelatin-coated well of the plate, and incubate it at 37 degrees Celsius for a minimum of 24 hours. Then on day six of culture, aspirate the spent medium from the transduced cells and wash them in 1X PBS. Next, add one milliliter of 0.05%trypsin solution to each well of the plate.
After the cells are detached, proceed to neutralize with trypsin neutralizing solution or TNS, then collect and centrifuge the cells as previously discussed. Following centrifugation, aspirate the supernatant, and resuspend the cells in four milliliters of BEGM, supplemented with penicillin and streptomycin. Proceed to introduce two milliliters of this cell suspension into each of gelatin-and MEF-coated wells of the previously prepared plate.
After incubating the cells overnight, replace the BEGM with 2.5 milliliters of standard human embryonic stem cell, abbreviated as hESC, medium, containing four nanograms per milliliter basic fibroblast growth factor. Proceed to culture the cells and feed them daily with fresh hESC medium until hESC-like colonies, harboring putative induced pluripotent stem cells, or iPSCs, are observed in the cultures. Manually excise, and transfer these colonies to separate wells of a 24-well plate in order to culture and expand them as separate lines in a feeder-free system.
Initially, nasal samples are a mix of cell types and debris. However, following culture and medium changes, epithelial cells become predominent in these preparations, as shown in this picture take one day after cell passaging. Here, epithelital cells, growing to confluence, demonstrate large nuclei and stretched cytoplasm.
Due to the properties of the lentiviral particles used in this study, when such nasal epithelial cells are transduced, they express the tdTomato fluorescent marker, as demonstrated by the upper right image of cells, four days post-transduction. A comparison with the corresponding bright-field picture reveals that not every cell demonstrates tdTomato signal as evidenced by the merged image on the bottom. Thus, not every cell is transduced.
However, transduction efficiency is approximately 90%When transduced nasal epithelial cells are cultured as described in the protocol, they give rise to iPSC colonies. Following iPSC colony harvest, and culture in standard feeder-free conditions, individual cells in such colonies demonstrated typical hESC-like morphology, such as a round shape, large nucleus, and high nucleus to cytoplasm ratio as demonstrated by this magnified image. Once mastered, this technique can be done in one and a half hours, if performed properly.
When attempting this procedure, it's important to remember to take the sample correctly, keep the cells at 37 degrees Celsius, and plate them as quickly as possible at the correct density. Following this procedure, other methods like setting up an air-liquid interface using primary nasal epithelial cells can be performed in order to answer additional questions like how the air we respond to allergens and pathogens. After its development, this technique paved the way for researchers to model diseases such as asthma, COPD, and cystic fibrosis.
This technique also helps study environmental effects on lung development in the in vitro human system. After watching this video, you should have a good idea of how to cultivate nasal epithelial cells from children and generate iPSCs from this population. Since we work with human embryonic stem cells and induced pluripotent stem cells, remember to always adhere to institutional and national guidelines.
