The overall goal of this three-dimensional model of human precision-cut lung slices is to asses cytotoxicity and immunomodulatory effects in an ex vivo model using human tissue. These methods can help answer key questions in the field of pharmacology and toxicology in order to evaluate safety and help to asses physiological response to substances in the native human environment. The main advantage of this technique is that we are able to mimic human respiratory diseases regarding cytotoxicity or immunomodulatory effects, thanks to the usage of fresh, viable human tissue.
Though this method can provide insight into human cell-cell interactions, physiology, and pathomechanism, in respiratory disease, it can also be used for other applications as well as for other species. Generally, individuals new to this method will struggle with the organization of assessing human tissue and establishing a reliable infrastructure. Moreover, the filling process requires lots of training and experience, due to the heterogeneity of the lungs.
To begin this procedure, cannulate the trachea by inserting the silicone tube and fixing it with a clamp parallel to the tube so that the clamp squeezes the tissue together alongside the silicone tube without pinching it off. Close all other bronchi, blood vessels, and injuries with clamps so that no agarose can leak out during the filling procedure. Good anatomical skill are needed to cannulate the main bronchus.
The lungs should not be expanded too much, otherwise the tissue will distract, and if the tissue is not filled enough, it will be impossible to make any PCLS because the tissue is too soft. Then, mix an equivalent volume of 3%low-gelling agarose with culture medium in a beaker. Instill the mixture into the lung using a 15 milliliter syringe.
Prior to refilling the syringe with medium, clamp shut the catheter with fingers or a clamp to avoid air bubbles and agarose reflux. Cut the lung tissue into three to five centimeter slabs. Fill the tissue slicer with 400 milliliters of ice cold EBSS.
Immediately cut the cylindrical tissue cores out of the lung slabs, using a semi-automated screwdriver with a coring tool. Afterward, transfer the tissue cores into the tissue holder of the tissue slicer. Place the weight on top of the tissue core and start slicing the tissue core into PCLS.
Medium should be drained out of the tissue slicer into a beaker, by opening the clamp of the glass cylinder. Next, transfer the slices from a beaker to the Petri dish with culture medium. Following that, place the Petri dish into an incubator and allow the medium to warm up prior to the washing steps.
Then, transfer the lung slices carefully into a 24-well culture plate with a minimum of 500 microliters of culture medium for two slices per well. To prepare a master mix of the working solution, dilute 25 microliters of the WST-1 Reagent and 225 microliters of medium for each well. Next, pipette 250 microliters of the master mix working solution of WST-1 for each well and incubate the plate at 37 degrees Celsius for one hour.
Ensure that the PCLS are fully covered by the WST-1 Reagent during incubation. Afterward, place the plate on a and orbital shaker at 200 RPM, and shake carefully for 30 seconds to ensure thorough mixing of the WST-1 Reagent. Then, pipette 100 microliters of the supernatant from each well of the 24-well plate to a new, flat-bottomed, 96-well plate in duplicates.
Measure the absorption of each well at 450 nanometers, using a microplate reader and subtract the absorption at 630 nanometers reference from that at 450 nanometers. After incubating the PCLS with or without test agents, transfer 50 microliters of supernatant in duplicates into a new, 96-well plate. This generates duplicates from each treated well of a 24-well plate.
Immediately before the assay, prepare a master mix of the working solution of LDH-free agent, by thoroughly mixing 125 microliters of catalyst solution with 6.25 milliliters of dye solution for a 96-well plate. Then, pipette 50 microliters of the master mix working solution into each well already containing 50 microliters of supernatant. Incubate the plate for 20 minutes at room temperature in the dark.
Afterward, measure the absorption of each well at 492 nanometers, using a microplate reader, and subtract the absorption at 630 nanometers reference from that at 492 nanometers. For microscopic assessment, perform at least two randomly distributed Z-stacks, with the cLSM. Click on the Ocular tab to choose a 10-fold objective.
Click on Online to use the cLSM as a standard light microscope to find the surface of PCLS. Then, click Offline to exit the ocular setting. Subsequently, click the Acquisition tab and turn on the appropriate lasers for the fluorophores.
Click on Light Path under the Acquisition tab, and set up the necessary filters and mirrors for the experiment. Press the live button to see a live view of the corresponding layer on the screen. Move the focus up or down to find the surface of the PCLS with a sharp signal.
Subsequently, set the pinhole for the red ethidium homodimer-1 channel to one air unit for the best trade off between efficiency of light collection and optical sectioning, and adjust the calcein channel accordingly. Increase the detected signal of the gain. Increase or decrease the digital offset to adjust the background such that appears in blue in the live image with activated channel color lockup table.
Check the Z-stacks box to set the upper and lower limits for the microscopic volume. Slowly shift the focus up or down until the range of 30 micrometers has been reached and press Set Last to save. Then, click on Live once again to deactivate the live image, and press on Start Experiment to start the imaging.
Shown here, are the microscopic viability images of PCLS, which demonstrates the responsiveness of the human tissue to detergent as an effective toxic substance. Toxic effects are visually assessed by evaluation of calcein positive tissue compared to ethdium homodimer-1 positive cell nuclei. Even though the viability of human lung tissue varies among donors, the number of dead cell nuclei compared with viable tissue should not exceed 15%of the tissue control.
This figure displays the representative data for the immunomodulatory effect of ammonium hexachloroplatinate, an SLS on human lung tissue. Extracellular and intracellular IL-1 alpha and TNF-alpha were determined by ELISA and normalized to the total protein content. After its development, this technique paved the way for researchers in the fields of pharmacology and basic science to explore physiological responses within viable human lung tissue.
Following this procedure, other endpoints like airway constriction can be performed in order to answer additional questions, such as bronchodilatory drug efficacy. After watching this video, you should have a good general understanding on how to generate and process human precision-cut lung slices. Once mastered, this technique can be done within six to eight hours with the first results being available within the first 24 hours, if performed properly.
However, don't forget that working with human non-fixated material can be pu-tid-if-ly infectious, and precautions, such as protective clothing, should always be worn while performing this procedure. Additionally, it's important to remember to work under sterile conditions as much as possible.
