The overall goal of this procedure is to isolate airway epithelial cells in order to study the toxic effects of cigarette smoke exposure. This is accomplished by first dissecting mouse tracheas and then digesting the trachea's in pronase solution. The second step of the procedure is to collect epithelial cells and propagate the quid culture.
The third step is to differentiate the cultures at an air liquid interface. The final step of the procedure is to expose epithelial cells to apical cigarette smoke at the air liquid interface. Ultimately, results can be obtained that show a fully differentiated monolayer of epithelial cells by immunofluorescence microscopy.
This method can help answer key questions in the chronic obstructive pulmonary disease field, such as the effects of cigarette smoke exposure on CLIA morphology and cigarette smoke induced biochemical markers of cell injury. Begin preparations for this procedure one day prior to the dissection by preparing hams. F 12 media containing antibiotics hams F 12 media containing antibiotics and 20%F-B-S-M-T-E-C, basic medium containing antibiotics.
MTEC medium with 5%FBS DNA's one solution retinoic acid stock solutions and MTEC proliferation medium according to the instructions in the written protocol. Sterilize all surgical tools to be used in the dissection on the day of the dissection. Ensure that the dissection area is clean and that all dissection tools are at hand.
Sterilize the tissue culture hood. Then working in the tissue culture hood. Prepare a solution of 0.15%pronase.
Add 15 milligrams of pronase to 10 milliliters of hams. F 12 media containing antibiotics in a 50 milliliter conical tube. Place on ice until use.
Mouse tracheas are dissected from mice Using standard surgical procedures, disinfect the tube containing the tracheas and place in the tissue culture hood. Transfer the trachea tissue to a 100 millimeter Petri dish with hams F 12 media containing antibiotics. Use sterile surgical forceps to remove the connective tissue from the tracheas.
Then transfer the clean tracheas into the second 100 millimeter Petri dish containing hams. F 12 media with antibiotics. Use dissection scissors to cut the tracheas along the vertical axis and expose the lumen.
Transfer the tracheas to the 50 milliliter conical tube containing 10 milliliters of Pronase solution and place in the refrigerator to incubate overnight at four degrees Celsius. Finally, prepare collagen one treated transwell plates for use. The following day, add 50 milligrams of collagen, one per milliliter of 0.02 N acetic acid pipette one milliliter of this solution into each well of a 12.
Well trans well plate. Wrap the plate in para film and let's stand overnight at room temperature in the tissue culture hood. The next day.
Gently invert the tube containing the mouse tracheas 10 to 12 times. Then allow the contents of the tube to incubate for an additional 30 to 60 minutes at four degrees Celsius. Working in the tissue culture hood pipette 10 milliliters of hams F 12 media containing antibiotics in 20%FBS into the conical tube containing the digested tracheal tissue and invert 12 times.
Then pipette 10 milliliters of hams F 12 media containing antibiotics in 20%FBS into each of three 15 milliliter conical tubes. Use a sterile pasta pipette to transfer the tracheal tissue from the pronase solution to the first of the tubes containing supplemented hams. F 12 media.
Set the Pronase solution on ice cap the tube containing the tracheal tissue and gently invert 12 times to mix. Then using the pasta pipette, transfer the tracheal tissue to the second 15 milliliter conical tube and invert this tube 12 times. Repeat the transfer and inversion procedure using the final 15 milliliter conical tube.
Finally, use the pasta pipette to remove and discard the tracheal tissue. Then retrieve the pronase solution from ice and combine the three 15 milliliter volumes of supplemented hams F 12 media with the pronase solution. Centrifuge the sample in the chilled centrifuge at four degrees Celsius for 10 minutes at 1, 400 rotations per minute following centrifugation discarded the supena, then pipette approximately 100 to 200 microliters of DNA's, one solution per trachea onto the pellet and reus.
Suspend with gentle tapping. Incubate the cells on ice for five minutes. Centrifuge the tube at 1, 400 rotations per minute for five minutes of four degrees Celsius and discard the supinate.
Then pipette eight milliliters of MTEC medium containing 10%FBS onto the pellet and resuspend plate. The cell suspension on a paria plate incubate at 37 degrees Celsius in an atmosphere of 95%air and 5%carbon dioxide for five to six hours. This is a negative selection step for the removal of fibroblasts.
Following incubation, collect the cell suspension from the paria plate and pipette into a 50 milliliter conical tube. Then rinse the plate twice with four milliliters of MTEC medium containing 10%FBS. Transfer both of the washes to the 50 milliliter conical tube containing the cell suspension.
Pipette one milliliter of the cell suspension into an einor tube. Reserve this aliquot for cyto spin analysis and cell counting centrifuge. The 50 milliliter conical tube containing the remaining 15 milliliters of cell suspension are 1, 400 rotations per minute at four degrees Celsius for 10 minutes.
During the centrifugation, remove and discard the collagen solution from the previously prepared transwell plate. Leave the plate to dry under the hood for five minutes, then wash twice with PBS after the final wash. Aspirate all of the PBS and pipette 1.5 milliliters of proliferation media into the basal compartment of the trans well following centrifugation.
Discard the supinate and resuspend the cell pellet in an appropriate volume of proliferation media to facilitate plating of 7.5 times 10 to the four to one times 10 to the five cells in 500 microliters of suspension per well. Pipette 500 microliters of cell suspension onto the apical surface of the transworld polycarbonate membrane.Insert. Incubate the submerged MTEC cultures at 37 degrees Celsius in a humidified incubator containing 95%air and 5%carbon dioxide for seven to 10 days with media changes every other day.
Measure trans epithelial cell resistance using an intervalometer. When epithelial resistance reaches 1000 ohms per centimeter squared and the cultures appear confluent. Convert to a LI culture by removing the apical media.
Also replace basal media with MTEC basic media containing 2%new serum and retinoic acid. Incubate the cultures at 37 degrees Celsius and 5%carbon dioxide for 10 to 14 days to allow differentiation with media changes every other day. First, set up the smoking machine according to the manufacturer's instructions.
Record the weight of an unused pole flex filter. Then place the filter in the inline stainless steel holder on the sampling tube leading from the sampling port on the smoking chamber to the constant flow pump. Connect the sampling unit to the gas meter using an outflow tube using a CS cell exposure system.
Expose the apical side of the transworld cultures to mainstream cigarette smoke from three R four F research reference filter cigarettes. Expose the cells to the smoke from one to two cigarettes. Expose control cultures to room air for an equivalent exposure period after gas sampling gain weight.
The pool flex filter, the total material deposited on the filter in milligrams is normalized. For the total volume of air sampled the smoke from one to two cigarettes typically yields 100 to 200 milligrams per cubic meter of TPM. Post cigarette exposure, harvest the cells and basal media for further analyses.
This is an example of a healthy monolayer of mouse respiratory epithelial cells stain stained for nuclear epithelial cell, and C markers. Successful epithelial isolation, proliferation and differentiation uses an intact monolayer with a cobblestone morphology. The cultures are essentially fibroblast free.
These electron micrograph images are representative of healthy MTEC cultures and depict the ultra structure of the various cell types and CELIA morphology. This live cell imaging video shows healthy C morphology of MTEC cultures. After watching this video, you should have a good understanding of how to isolate mouse tracheal epithelial cells, differentiate them in an air liquid interface, and expose them to mainstream cigarette smoke.
