The PCLS supports the assessment of airway and vascular smooth muscle cell activity in a nearly intact tissue environment, thereby providing an invaluable ex vivo model for pulmonary research. In term of smooth muscle research, the precision-cut lung slices preserve the in vivo phenotype of smooth muscle cells and their interaction with surrounding structures while allowing access to smooth muscle cell, which is important for a mechanistic study at the cellular level. To begin, place the mouse body on a dissection board in the supine position.
Pin down the tail, front paws, and head with 25G syringe needles and sanitize the body with 70%ethanol. Open the chest cavity carefully along the sternum and bilateral inferior rib cage above the diaphragm. Then, point the sharp scissor tip away from the lung tissue and remove part of the bilateral ventral rib cages to expose the heart.
Observe that the lung lobes collapse when the chest cavity opens. Use scissors to remove soft tissue in the mouse neck to expose the trachea. At the upper end of the trachea make a small hole of diameter 1.2 millimeters that should allow the passing of a 20G Y-shaped IV catheter tip.
Connect one adapter port of the Y-shaped catheter with a 3 milliliter syringe prefilled with 0.5 milliliters of air and the other port with a 3 milliliter syringe prefilled with 2 milliliters of 1.5%agarose solution warmed to 42 degree Celsius. Inject agarose solution to fill the catheter and then push the catheter through the opening into the trachea to 5 to 8 millimeters. Slowly inject the agarose solution at a rate of 1 milliliter per 5 seconds.
Observe that the lung expands along the proximal to distal axis. Stop the injection when the edge of each lung lobe is inflated. Then, inject 0.2 to 0.3 milliliters of air from the other syringe to push the residual agarose into conductive airways to the distal alveoli space.
Clip closed the trachea with a pair of curved hemostatic forceps. To infill the pulmonary vasculature, fill a 1 milliliter syringe with warm 6%gelatin and connect it to a needle scalp vein catheter. Infill the catheter with gelatin solution and then puncture the right ventricle close to the inferior wall with the needle.
Push the needle for 2 to 3 millimeters into the right ventricle and point the needle tip to the main pulmonary artery. Inject approximately 0.2 milliliters of gelatin solution slowly into the right ventricle and pulmonary arterial vessels. Keep the needle in place for five minutes after injection and cool the lung lobes by pouring ice cold HBSS solution on the heart and lung and place the body in a refrigerator.
After this step, remove the mouse lung and heart from surrounding connective tissues with scissors. Then, separate each lung lobe and keep them in HBSS solution on ice. Trim the lung lobe and orient it such that the cutting direction is perpendicular to most airways from the hila to the lung surface.
Attach it on top of the specimen column with super glue. Use a vibratome with a fresh thin razor blade to cut the lung lobe into 150 micrometer slices. Collect the slices in sterile Petri dishes prefilled with cold HBSS solution.
Transfer the slices to Petri dishes filled with DMEM/F-12 culture medium. Place the dishes in a 37 degree Celsius incubator overnight before the experiments. Place a single precision-cut lung slice in each well of a 24 well culture plate filled with HBSS.
Locate the slice in the middle of the well and then remove the HBSS solution with a pipette. Under a microscope, find the target airway and vessel in the slice and then cover it using a nylon mesh with a precut central hole to expose the targeted airway vessel area. Put a hollow metal washer on top of the mesh to hold the slices in place.
Then, add 600 microliters of HBSS solution to submerge the slices. After 10 minutes, record the baseline images. To induce the airway or vascular contraction, cautiously remove the HBSS solution with a pipette and add 600 microliters of HBSS with an agonist.
To prepare the calcium dye loading buffer, first dissolve 50 micrograms of dye with 10 microliters of DMSO and 0.2 grams of Pluronic F-12 powder in 1 milliliter of DMSO. Mix 10 microliters of the Pluronic solution with 10 microliters of calcium dye solution. Add this mixture to 2 milliliters of HBSS solution containing 200 micromolar of sulfobromophthalein.
Then, place 15 precision-cut lung slices in 2 milliliters of calcium loading buffer and incubate at 30 degrees Celsius for one hour, followed by incubating in HBSS containing 100 micromolar sulfobromophthalein for 30 minutes. Then, place calcium dye loaded slices on a large cover glass. Fill high vacuum silicone grease in a 3 milliliter syringe attached to 18G blunt needle or a 200 microliter pipette tip and draw two parallel lines across the cover glass, above and below the slice.
Cover the slice using an nylon mesh between two grease lines. Place the second cover glass on top of the mesh to generate a chamber. Add HBSS or a agonist solution into the chamber from one end with a pipette.
Remove the fluid from the chamber by suctioning from the other end with tissue paper. The slice's chamber is now ready for the calcium imaging with a high speed laser scanning confocal microscope. Pulmonary airway artery bundles were observed in precision-cut lung slices of thickness 150 micrometers under a phase contrast microscope.
At resting state, an airway was identified by cuboidal epithelial cells with a nearby pulmonary artery. Following exposure to methacholine, the luminal area was reduced while the pulmonary artery showed no response to the stimuli. Airway contractile responses were quantified by the percentage of luminal area reduction and showed similar dose dependent responses in one day and five day cultures.
Upon reaching the peripheral lung field, the conductive airways branch into respiratory ducts and sacs surrounding the small intra-acinar arterioles. When exposed to endothelii both airways and pulmonary arteries constrict, which is followed by NOC-5 induced relaxation. At the resting state, the calcium dye loaded slices showed low fluorescence in the smooth muscle cells of the airway and vascular system under a confocal fluorescent microscope.
Upon exposure to agonists, the calcium fluorescence intensity elevated in the cells and propagated to the entire cell, which correlated with the oscillatory signals. Technique wise, it is essential to inflate the lung with agarose homogeneously and avoid quick or excessive agarose injection. Always push air at the end to flush the agarose from the conductive airway to the distal alveoli space.
In summary, using the precision-cut lung slices culture, one can provide a special variety of pulmonary smooth muscle functions and model the smooth muscle deregulation in vitro. It also provides an ideal platform to screen novo vasodilatory or bronchodilator medications.
