The overall goal of the following experiment is to perform long-term co-culture of Nontypeable Hemophilus influenza or NTHI with primary human respiratory epithelial tissues using the epi airway model. This is achieved by inoculating the apical surface with NTHI to allow the bacteria to adhere to and enter the tissues as a second step. The inserts are washed every 24 hours with prewarm DPBS to mimic normal mucus flow and to remove mucin waste products and any cellular debris.
Next, the tissues are harvested at the desired time point and the survival of either total cell associated or internalized NTHI is determined. Finally, with this method, strain specific characteristics of NTHI survival within the tissues over time based on viable bacteria counts can be evaluated. Though this method can provide insight into long-term infections with nontypeable hemophilus influenza.
It can also be used in the study of other human pathogens such as Moraxella Alis demonstrating this procedure will be Dr.Doin ran a postdoctoral fellow in my laboratory. Remove the transport plate from its packaging and prepare to begin the experiment. Spray the transport plate with 70%ethanol and place the plate in the biosafety cabinet.
Aliquot one milliliter of four degrees Celsius, epi airways, maintenance media into each well of a six well plate. Then after removing the tape, open the plate and remove the gauze covering the epi airway inserts. Using a twisting motion, use tweezers to release an insert from the aros.
Place the insert into one of the wells of the six well plate If needed. Use a sterile cotton swab to carefully remove the aros from the insert, avoiding touching the membrane. Confirm that no air bubbles are trapped beneath the inserts.
And then place the six well plate in an incubator. Wash the inserts daily as follows, prewarm DPBS with calcium and magnesium, and fill a tip with the DPBS. Then using sterile forceps, pick up an insert gently pipette 200 microliters of the prewarm DPBS onto the tissue.
Then rock the insert to evenly distribute the DPBS without touching the tissue. Next, tilt the insert at an angle and place a P 1000 pipette tip against the plastic ring that holds the membrane at the bottom of the insert. Then carefully aspirate the buffer solution and place the solution into a cryo tube for later study.
Finally, aspirate the basal maintenance media and replace it with one milliliter of fresh media. Prewarm more DPBS with calcium and magnesium. Add three milliliters of the prewarm DPBS to a 15 milliliter tube, and then resuspend single colonies of nontypeable influenza or NTHI grown overnight on chocolate agar into the tube.
Now vortex the tube vigorously for at least 10 seconds, and then measure the optical density. Continue to add single colonies of the NTHI until the bacterial concentration reaches an OD 600 nanometers of approximately 0.7. Next, wash each EPI airway insert with prewarm DPBS as before, and then add 28 microliters of the bacterial suspension to the apical surface of each insert.
Avoiding inoculating the basal maintenance media. Then return the plate to the incubator to quantify the NTHI first prewarm, and then surface dry chocolate agar plates. Next serially dilute the NTHI inoculum using sterile PBS with 0.1%gelatin or PBSG.
Drop 10 microliter aliquots of the inoculum onto the plates, and then incubate the plates overnight at 37 degrees Celsius in 5%carbon dioxide. The next day, determine the CFUs per milliliter by counting the dilution that have 15 to 30 distinct colonies in each drop, and calculating back to your actual viable inoculum of NTHI to harvest the total cell associated bacteria. Rinse the apical surface of the epi airways three times with 200 microliters of DPBS without calcium or magnesium.
Then tilt the insert and rinse any remaining maintenance media from the basal membrane. Place the washed insert in a fresh six well plate and add 250 microliters of sterile, 1%saponin solution onto the apical surface of each tissue. Return the plate to the incubator for 10 minutes while the plate is incubating.
Set up tubes for serial dilution filling each dilution tube with 900 microliters of PBSG. Next, scrub the tissue from the membrane with a P 1000 pipette tip using a back and forth motion, followed by a circular motion to remove the tissue from the edges of the insert. Then using a sterile large bore P 1000 pipette tip, place the suspension into an empty 1.5 milliliter einor tube.
Now add 250 microliters of DPBS onto the apical surface of the insert, and then examine the insert using an inverted phase contrast microscope to determine whether any tissue remains to be harvested. Add the suspension to the einor tube and top with 500 microliters of DPBS vigorously. Vortex the cell suspension for one minute.
Then using a sterile one milliliter syringe fitted with a 26 gauge needle. Aspirate the suspension into the syringe through the needle. Slowly pass the suspension through the needle three times, taking care not to create bubbles.
Then vortex the suspension vigorously for another minute. Next, use a large bore pipette tip to transfer 100 microliters the suspension into the first dilution tube. Vortex the tube vigorously for five seconds, and then make one to 10 serial dilution drop plate the dilution onto chocolate agar plates.
Then incubate the plates overnight at 37 degrees Celsius in 5%carbon dioxide for viable bacterial counts to harvest internalized bacteria. Only wash each insert three times with 200 microliters of DPBS. Replace the basal maintenance media with media containing 100 micrograms per milliliter gentamycin sulfate, and then add 300 microliters of the Gentamycin containing maintenance media onto the apical surface.
Return the inserts to the incubator for one hour while the inserts are incubating prewarm some DPBS. Then remove the Gentamycin containing maintenance media from the apical surface. Next, wash the apical surface and basal membrane of the inserts extensively with the prewarm DPBS.
Finally, repeat the scrubbing and harvesting procedure just demonstrated before performing serial dilutions and plating of the internalized bacteria in this figure. Comparing scanning electron microscope images of uninfected epi airway tissues on the left with five day NTHI infected epi airway tissues on the right. It can be seen that long-term co-culture with NTHI does not result in significant damage to the apical tissues, underscoring the utility of the epi airway model.
Here a graph depicting the number of NTHI internalized over time during an epi airway Co-culture is shown. The R 2 8 6 6 strain was inoculated at approximately one times 10 of the seventh CFU per insert. Then internalized bacteria were harvested and enumerated at each indicated time point.
After watching this video, you should have a good understanding of how to perform long-term co-culture of NTHI with the primary hormone respiratory tissues using the A PRE model.
