The overall goal of this procedure is to establish a polarized liquid covered culture of Calu three cells using a Transwell culture system to set up the Transwell culture system. First, prepare a transwell plate by moving inserts into exterior rows of the plate and adding medium to the basolateral chambers of each well. The second step is to prepare mono layered culture cells into single cell suspensions.
Next, the suspended cells are seated into the apical compartments of the transwell inserts. The final step is to completely replace apical and basal lateral media. In culture inserts on a three and then four day cycle for two to three weeks until cells are fully polarized.
Ultimately, trans epithelial electrical resistance and a passive sodium fluorescein equilibration assay are used to demonstrate polarization of liquid covered calu three cultures. The main advantage of using this technique over using normal human bronchial epithelial cells is that Calor three are more accessible and develop more rapidly into polarized cultures Ready for use All procedures for growing polarized calu. Three liquid covered cultures or LCC are performed in a biosafety cabinet using sterile culture techniques.
Begin by preparing a 24 well trans well plate. For seeding using sterile forceps, move transwell inserts from the interior to the exterior rows of the plate without touching the insert. Membrane cells should only be subculture into wells on the exterior rows of the plate.
At 600 microliters of EMEM containing 10%FBS referred to hereafter as EMEM 10%to each basolateral compartment. To prevent the introduction of air bubbles, angle the pipette against the wall of each compartment and slowly release the medium into the well. Next detach Colu three cells from tissue culture flasks and subculture cells as described in the protocol text thoroughly resuspend the washed cells in five milliliters of EMEM 10%by gently pipetting up and down, determine viable and total cell counts by the trian blue exclusion method and proceed only if 80%to 90%are viable.
Then in a 50 milliliter sterile conical tube dilute cells to a concentration of two times 10 to the six viable cells per milliliter. With EMEM 10%add 100 microliters of resuspended calu three cells to the apical compartment of each transwell except wells A one and D one. Gently angle the pipette against the interior guide channel of the insert wall and slowly release the cells.
Do not touch the pipette to the insert membrane. Gently agitate the CALU three cell suspension during this seating step to maintain a homogeneous suspension of cells to wells A one and D one, which will be cell-free control wells or blanks at 100 microliters of EMEM 10%without cells. Incubate the trans well plate at 37 degrees Celsius and 7%carbon dioxide in air atmosphere in an incubator where physical disturbance will be minimal to improve the efficiency of polarization.
Do not stack plates on top of each other three days after subculture and calu three cells into transwells, replace the medium completely gently aspirate medium from the transwell inserts by using a capillary pipette attached to a vacuum trap with a gentle vacuum. Remove apical medium from all wells first and then remove basolateral medium from all wells. Do not touch the insert membranes while aspirating medium.
Add 200 microliters of EMEM 10%to the apical compartment of cell-free wells A one and D one. Then to the seated wells, directing the medium into the apical compartment using the side of the insert to guide the pipette tip. Do not add medium directly onto cells and do not touch the insert membrane.
Next, add 600 microliters of EMEM 10%to the basal lateral compartments to prevent the introduction of air bubbles into the basal lateral compartments. Add medium to each one by angling the pipette against the wall of each compartment and slowly releasing the medium into the well. Return the transwell plate to the incubator after this first medium.Replacement.
Medium should be replaced on a cycle alternating between the fourth and then third day after the previous feeding until cells are fully polarized. Polarization is monitored by evaluating trans epithelial electrical resistance or tear of the CALU three LCC using a volter that has been calibrated and tested. To begin this procedure, remove the electrode from the ethanol air dry for five to 10 seconds and rinse the electrode with sterile EMEM 10%Set the mode switch of the volter to the resistance setting and turn the power on.
Begin measurements with the cell-free control wells to obtain baseline measurements and then continue with measurements for each. Well gently place the electrode into one of the three ports that allow access into the basolateral compartment of the transwell culture. Place the electrode so that the longer lead just lightly touches the bottom of the outer well and remains vertical.
And the shorter lead is in the tissue culture medium of the apical compartment without touching the insert membrane, push the measure R button and wait for the reading to stabilize. Repeat for the other two ports for each well and record the measurements from all three ports for a total of three measurements per well to verify that polarization is complete. A secondary assay measuring passive sodium fluorescein diffusion between the apical and basolateral compartments is performed on one to three transwell cultures.
Carefully remove medium from both the basal lateral and apical compartments of the transwell cultures that will be tested. Rinse transwell cultures by gently adding 600 microliters of room temperature, sterile DPBS to the basolateral compartments and 100 microliters of room temperature sterile DPBS to the apical compartments. Gently remove DPBS from the wells.
Add 600 microliters of sterile non fluorescent buffer to the basolateral compartments, followed by 100 microliters of sterile sodium fluorescein to the apical compartments. Incubate the plate at 37 degrees Celsius for one hour. After one hour.
Transfer the non fluorescent buffer sample from the basolateral compartment of each test transwell to a separate clean tube. For analysis, remove the test transwells used to examine passive sodium fluorescein diffusion from the plate and discard. Return the plate with remaining transwells to the incubator.
Place 100 microliters of each previously prepared sodium fluorescein standard solution in triplicate into a 96 well flat bottom plate. Prepare three dilution of each basal lateral sample in non fluorescent buffer. For each sample, add 100 microliters of an undiluted sample and 100 microliters of each dilution in duplicate into a 96 well flat bottom plate.
Measured the sample absorbance on an EISA plate reader at 486 nanometers or 490 nanometers. To begin this procedure, first wash cells and serum free EMEM with a capillary pipette attached to a vacuum trap with a gentle vacuum gently aspirate medium from all wells, removing the apical medium followed by the basolateral medium, aspirate medium from cell-free wells A one and D one, and then from seeded wells. Do not touch insert membranes while aspirating medium.
Add 100 microliters of serum free EMEM to the apical compartment of cell-free wells A one and D one, and then to the seated wells directing medium into the apical compartment. Using the side of the insert to guide the pipette tip. Do not add medium directly on cells and do not touch the insert membrane.
Next at 600 microliters of serum free EMEM to the basolateral compartments. Add medium to each trans well by angling the pipette against the wall of the compartment and slowly releasing the medium into the well Gently aspirate serum free medium from wells. Beginning with uninfected or mock infected wells, add the following virus dilutions to the apical compartment of the appropriate trans wells.
100 microliters of serum free EMEM for uninfected wells. 100 microliters of virus free preparation diluted in serum free EMEM for mock infected wells and 100 microliters of virus diluted in serum free EMEM for virus. After that, at 600 microliters of serum free EMEM to all basolateral compartments, incubate at 37 degrees Celsius and 7%carbon dioxide for two hours.
After two hours, aspirate medium from wells first from uninfected and mock infected wells, and then from infected wells. Remove apical supernatants first followed by basal lateral supernatants. Finally, replace medium with 200 microliters of EMEM, 10%in the apical compartments and 600 microliters of EMEM 10%in the basolateral compartments following the method demonstrated in this video.
The trans epithelial electrical resistance or tear of calu three liquid covered cultures reaches a plateau at or above 1000 M by centimeter square within three weeks after seeding. An example of which is shown in this figure since the tight junctions formed between polarized cells prevent passive equilibration of small molecules between the apical and basolateral compartments. A modified sodium fluoresce equilibration assay is used to confirm polarization of calu three liquid covered cultures.
As the tier of calu three cell monolayers and liquid cultures increases the amount of fluorescein that passively equilibrates into the basolateral compartment decreases cumulative data from four independent experiments is presented here, and each data point represents an individual measurement. Once the tear is 1000 owned by centimeter square, the amount of fluorescein that equilibrates into the BA basolateral compartment is less than or equal to 1%Therefore, calu three liquid covered cultures are considered to be fully polarized when the tier is greater than or equal to 1000 owned by centimeter square. Once the tier of calu three liquid covered cultures plateaus at or above 1000 owned by centimeter squared, the model is ready to be used to examine airway epithelial cell responses to respiratory pathogens, including respiratory syncytial virus or RSV as shown.
In this example, exposure to RSV represented in red results in a more rapid decline in polarized culture. Integrity compared to a mock infection of cells represented in blue data are presented as median resistance of eight independent wells per infection, plus or minus SEM per time point. The asterisk indicates P less than 0.05 between mock and RSVA two infected cultures as determined by the student's T-test test.
Don't forget when working with infectious agents, it can be hazardous and you should wear appropriate PPE and use good laboratory practices when performing these procedures.
