Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Precision cut lung slices method will preserve the structure and spatial orientations in three dimensions without regional bias and can serve as a live surrogate model for vessels and airway contractility measurements. In combination with time-lapse imaging, Western blot, and RNA analysis, precision cut lung slices contribute to the comprehensive understanding of signaling cascades that underlie a wide variety of disorders and lead to a better understanding of the pathophysiology in pulmonary vascular diseases. To begin, place the euthanized adult mouse in a supine position and secure it in place by taping the paws and nose to the table with adhesive tape.

Spray the ventral surface of the mouse with 70%ethanol. Use forceps to lift the abdominal skin of the mouse at the location of the bladder and surgical scissors to cut at the midline, moving superiorly to the cervical region to expose the trachea. Then use tweezers to lift the underlying fascia layer and use surgical scissors to expose the visceral organs and mediastinal compartment, again cutting inferiorly to superior.

Cut the sternum at the midline to expose the heart and lung. Dissect the diaphragm carefully from the liver and the abdominal compartment. Locate the inferior vena cava under the intestines and cut it.

After locating the right ventricle, use a 25 to 30 gauge butterfly needle to inject 0.5 milliliters of 1%fractionated heparin into the right ventricle and flush it slowly but steadily with 10 to 15 millimeters of PBS. After locating the larynx, use blunt tip tweezers to dissect the surrounding fascia and tissue, then place the surgical suture under the trachea and loosely tie a surgical knot. Place a small hole in the trachea superior to the string and cannulate with a 20 gauge blunt-ended needle.

Tighten the suture around the needle and trachea using a surgical knot. Then inject 2.5 to 4 milliliters of agarose into the trachea and monitor for inflation of the lung. After the agarose injection, pour cold PBS over the lung to solidify the agarose.

Then cut the trachea superior to the surgical suture and dissect the lung and heart from the mediastinum by removing any adhesions and fascia. After solidification, place the tissue in DMEM in a Petri dish on ice, and immediately proceed to slicing one lobe using a vibratome machine. To prepare the lung slices, attach the sample to the platform with superglue, keeping the medial side facing up.

Then fill the sample container with PBS. Fill the container with ice to keep the surrounding PBS and sample cold. Turn on the vibratome.

Adjust the thickness to 300 micrometers, frequency to 100 Hertz, amplitude to 0.6 millimeters, and speed to five micrometers per second. Using an Allen wrench, turn the blade holder into the safe position and open the jaws to insert a blade. Then tighten the jaws with an Allen wrench.

Place the sample onto the box, ensuring it is submerged in PBS. Slide the box up in the correct position and make sure it is secure. Turn the blade holder into the appropriate position for slicing.

Bring the vibratome blade up to the edge of the block and manually lower the blade until it is even with the top of the sample. Confirm the settings and run the vibratome. After the fresh slices are cut, remove the samples from the PBS and place them into a sterile Petri dish with 10 milliliters of DMEM containing one-time antimycotic antibiotic.

After placing all the slices in a Petri dish, retract the blade entirely and use the Allen wrench to raise the blade into a safe position. Store the samples in DMEM at 37 degrees Celsius. And place a vibratome slice on a microscope slide.

Use a cleaning wipe to remove excess medium. Place the sample on the phase contrast microscopy and use 10 to 20X magnification to identify a vessel. Then add 500 microliters of vasoconstrictors, such as 60 millimolar potassium chloride or one micromolar endothelin-1 to completely submerge the slide.

Observe and capture the video by recording for 30 to 60 seconds. Before beginning the experiment, fill a small polystyrene foam box with one liter of liquid nitrogen and place two 1.5 milliliter microcentrifuge tubes in the container with liquid nitrogen. Place four to five fresh vibratome slices in a mortar bowl.

Pour 5 to 10 milliliters of liquid nitrogen on top of the slices and quickly use the pestle to grind the slices into powder before the liquid nitrogen evaporates. Use a pre-chilled steel laboratory scooper to scoop the powder into the two chilled microcentrifuge tubes and lyse the powder by adding 500 microliters of RNA extraction reagent for RNA isolation or 500 microliters of RIPA buffer for protein lysis. In the representative viability experiment, the color change of the PCLS slices was detected from day zero to one and day nine to 10.

The solution was initially blue and turned pink overnight, demonstrating viability. Color change typically occurred within one to four hours, but a longer time may be necessary. The daily difference between absorbance at 562 and 613 nanometer wavelengths for the vibratome-prepared tissue is shown here.

The constriction of the vasculature in a vibratome-prepared lung tissue using endothelin-1 and potassium chloride can be visualized here. The preservation of tdTomato labeling in cells one week after tamoxifen injection is demonstrated in these lung slices. It is most important to perform the lung clearing and inflation step well to remove all blood from circulation before additional experiments and imaging.

In addition to vascular diseases, PCLS can be applied to study physiobiology of airway-related disease, and can be further tested on human lung tissues in clinically relevant scenarios.