Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

The overall goal of this procedure is to assess the impact of repeated exposure of Organotypic 3D bronchial and nasal tissue culture models to whole cigarette smoke. This is accomplished by first culturing organotypic, bronchial and nasal tissue culture models at the air liquid interface. The second step is to generate aerosol with a smoking machine and expose the tissue cultures according to the experimental design in an exposure system.

Next, the exposure impact is assessed by measuring the ciliary beating frequency and tissue integrity of the culture. The final step is to measure transcriptome exchanges induced by the aerosol using microarray technology. Ultimately, a network-based systems biology approaches used to show the impact of whole cigarette smoke on the tissue culture.

The main advantage of these techniques of existing methods in respiratory toxicology is that the approaches are aligned with regulatory agency requirements to find alternatives to animal testing, they enable the assessment of the effects of short-term cigarette smoke exposure on cells grown at the illiquid interface, mimicking the conditions occurring in the human respiratory tract. To begin the experiment, add 0.7 milliliters of prewarm culture media to each well of a sterile 24 well plate. Next, remove the tissue packaging and transfer the tissue culture inserts to the freshly prepared 24 well plate.

Maintain the tissue culture in an incubator and replace the media every two days during the media change. Examine the tissues under a light microscope to ensure they're contamination free and that there is no leakage of media. Three days before exposure experiments, wash the apical side of the tissue culture with 200 microliters of culture media.

Next, prewarm the climatic chamber of the in vitro system and the cultivation base module to 37 degrees Celsius. After it is warmed, fill the cultivation base module with 17.5 milliliters of media per row. Remove the tissues from the incubator and transfer the tissue culture inserts from the culture plate to the cultivation base module of the in vitro exposure system.

Then cover the cultivation base module with a glass lid. Transfer the tissues into the climatic chamber for exposure to mainstream cigarette smoke or cs. Generate cigarette smoke using a calibrated 30 port carousel smoking machine.

Smoke the cigarettes to a standard but length approximately 35 millimeters at 55 milliliters. Puff over two seconds, twice a minute, and eight second pump exhaust time. Mainstream cigarette smoke generated is accumulated and released to the in vitro exposure system.

Expose the tissues for six to seven minutes. After transferring the tissues back to a culture plate, place them back in the incubator for one hour. Remove the tissue culture plates from the incubator and leave them under the hood at room temperature for 30 minutes to stabilize the CLIA beating.

After stabilization, observe the tissues under a light microscope connected to the cliometrics software. Next, record the frequency of the ciliary beating, measuring the frequency every three seconds for one minute, 48 hours. After exposure, add 200 microliters of culture media to the apical side of the human bronchial epithelial tissues and measure the tissue resistance by inserting a chopstick electrode into the basal media and into the newly added media on the apical side of the tissue culture without touching the tissue.

After the measurements, gently remove the media from the apical side of the tissue cultures using a paster pipette connected to a vacuum pump and return the tissues to the incubator. To measure cytochrome P four 50 enzymatic activity, prepare a new plate with previously prepared Lucifer and CEE substrate and transfer the inserts into that plate cover and incubate the tissue inserts overnight. After the incubation transfer 50 microliters of culture media from each tissue insert into a 96 well opaque white luminometer plate at room temperature.

Then add 50 microliters of Lucifer detection reagent to each well and incubate the plate at room temperature for 20 minutes. Once incubation is finished, measure the luminescence with the luminometer. Collect the plate of 3D organotypic tissue inserts from the incubator and wash each insert on the plate three times with cold PBS and aspirate the PBS with a past pipette between washes.

After the last wash, aspirate the PBS and cover the plate to prevent the insert from drying. Next, cut out the insert membrane for each insert from the plate until the membrane lies down flat on the blade. Transfer the tissue from the blade to the homogenizing tube and screw the lid on the tube.

Then shake it, vortex it and place on dry ice. Defrost and grind samples with the homogenizing instrument at 6, 000 RPM for 45 seconds. Leave for five minutes at room temperature and then add 140 microliters of chloroform to the sample and vortex again for 15 seconds.

Transfer the whole sample from the homogenizing tube to a phase lock tube. Place it on a thermo shaker and shake it for two minutes at 1400 RPM at room temperature. Then leave for two minutes at room temperature.

Once shaking is complete, spin the tube for 15 minutes at four degrees Celsius at 12, 000 G.Transfer the supernatant to a new two milliliter tube. Follow with RNA precipitation wash, suspension, and purification. Once extracted, the RNAs are quality checked.

To assess quantity and integrity, prepare the liquid handling system and target synthesis. Place the extracted RNA. The plates and all necessary reagents and lab wears in the robot.

First RNA concentrations will be normalized. Then the deck will be changed for the next steps from amplification to labeling, purification, and fragmentation. Next, the hybridization mix is added to 33 microliters of the fragmented and labeled A RNA denature the samples and pre-wet the array with 200 microliters of pre hybridization.

Mix from the hybridization wash and stain kit. Then place the chip in the hybridization oven set at 45 degrees Celsius and 60 RPM for 10 minutes. Remove the pre hybridization buffer and load 200 microliters of the sample.

Incubate the array for 16 hours at 45 degrees Celsius and 60 RPM in the oven. Aspirate the cocktail from the chip and fill the microarray with 250 microliters of wash buffer a. Open the software and run fluidics from the software menu bar.

Select the module of interest and the prime four 50 program for each module. Assign the right chip to each module by scanning the barcode of the chip and place the chips in the fluidic system. Finally, ensure the scanner is warm.

Then load the chip into the auto loader of the scanner and scan the microarray. The CELIA beating frequency or CBF of nasal tissue was measured before and after exposure to air or cigarette smoke or cs. The CBF and nasal tissue was slower after exposure to cs.

A lower beating frequency of the CLIA reduces the efficiency to remove mucus and infectious agents. Microarray analyses were conducted to study the impacts of CS exposure in organotypic, bronchial and nasal tissue models. 48 hours after exposure.

This CS impact in vitro was compared to the impact of smoking on human bronchial epithelial tissue collected by bronchoscopy and nasal epithelial brushing respectively. The network-based systems biology shows a positive correlation between the backbone nodes of in vivo and in vitro data sets. While attempting this procedure, it is important to remember that this is a newly developing research area.

There are two key components on the one side, a well characterized toll aerosol exposure platform, and on the other side, a relevant experimental system which may consist of a three dimensional tissue culture model that best mimics the specific architecture, the biochemical and mechanical cues, as well as the cell cell interaction of the microenvironment relevant to the tissue or target organ. Under investigation.