The authors would like to thank Drs. Yuan Hao and Kaifeng Liu for their technical support. This work was supported by an NIH 1R01 HL150106-01A1, the Parker B. Francis Fellowship, and the Pulmonary Hypertension Association Aldrighetti Research Award to Dr. Ke Yuan.

All animal care was in accordance with the guidelines of Boston Children&#39;s Hospital and the Institutional Animal Care and Use Committee approved protocols. The mice used in this study are wild type C57/B6 mice and Cdh5-CreERT2 x Ai14 tdTomato crossed mice.
1. Preparation of solutions
<ol>
	<li>Prepare phosphate buffer solution (1x PBS) and 2% agarose solution required during the experiment in advance.
	<ol>
		<li>Mix 2 g agarose powder into 100 mL of autoclaved water. Heat it in the microwave a few seconds at a time until the solution is clear.</li>
		<li>Place the solution in a water bath at 42 &#176;C until use. 
		&#8203;NOTE: Both PBS and 2% agarose can be stored at room temperature for weeks.</li>
	</ol>
	</li>
</ol>
2. Extraction of the mouse lung
<ol>
	<li>Euthanize the mouse by isoflurane overdose. Achieve isoflurane overdose by placing a small amount of isoflurane on tissue paper in the lower level and placing the mouse in the upper level in a desiccator. The mouse remains in the desiccator until unconscious.</li>
	<li>Ensure death of the mouse by applying inferior pressure on the neck and pulling caudal on the tail. Bring the mouse to the dissecting surface.</li>
	<li>Place the mouse in the supine position and secure it into place taping paws and nose to table with adhesive tape.</li>
	<li>Spray the ventral surface of the mouse with 70% ethanol and wipe off the excess ethanol.</li>
	<li>Lift the abdominal skin of the mouse with forceps at the location of the bladder and cut at the midline with surgical scissors, moving superiorly to the cervical region, exposing the trachea.</li>
	<li>Lift the underlying fascial layer with tweezers and cut with surgical scissors to expose visceral organs and mediastinal compartment, again cutting inferiorly to superior.</li>
	<li>Cut the sternum at the midline to expose the heart and the lung.</li>
	<li>Using blunt dissection, detach the diaphragm from the liver and abdominal compartment.</li>
	<li>Locate the inferior vena cava (IVC) under the intestines and cut the IVC.</li>
	<li>Locate the right ventricle. 
	NOTE: The right ventricle will have a lighter appearance compared to the left ventricle and the intraventricular septum should be visualized, separating the right ventricle from the left ventricle.</li>
	<li>Inject 0.5 mL of 1% fractionated heparin to the right ventricle with a 25-30 G butterfly needle and then flush slowly but steadily with 10-15 mL of 1x PBS (Figure 1A).
	<ol>
		<li>Use caution not to insert the needle through the heart and inject heparin into the mediastinal cavity. 
		NOTE: The injection of 1x PBS turns the lung tissue white. The fluid extravasating from the abdominal aorta should be clear and colorless and the liver may become white in appearance after successful flushing.</li>
	</ol>
	</li>
	<li>Locate the larynx and dissect the surrounding fascia and tissue using blunt tip tweezers.</li>
	<li>Place the surgical suture under the trachea and loosely tie a surgical knot.</li>
	<li>Place a small hole in the trachea superior to string and canulate with 20 G blunt-ended needle.</li>
	<li>Tighten the suture around the needle and trachea using a surgical knot.</li>
	<li>Inject 2.5-4 mL of agarose into the trachea and monitor for inflation of the lung.</li>
	<li>After agarose is instilled, pour cold, chilled 1x PBS over the lung to solidify agarose.</li>
	<li>Cut the trachea superior to surgical suture and dissect the lung and heart from the mediastinum by removing any adhesions and fascia.</li>
	<li>After solidification, place in 1x DMEM (enough to submerge tissue) in a Petri dish to keep on ice. Immediately slice one lobe using a vibratome machine (Figure 1B).</li>
</ol>
3. Precision cut lung slices
<ol>
	<li>Attach the sample to the platform with superglue, keeping the medial side of the sample facing up.</li>
	<li>Fill the sample container with 1x PBS, ensuring that the sample is completely submerged.</li>
	<li>Fill the container surrounding the sample container with ice to keep surrounding PBS and sample cold.</li>
	<li>Turn on the vibratome and adjust to the desired settings below.
	<ol>
		<li>Set the thickness to 300um.</li>
		<li>Set the frequency to 100 Hz.</li>
		<li>Set the amplitude to 0.6 mm.</li>
		<li>Set the speed to 5 &#181;m/s.</li>
	</ol>
	</li>
	<li>Using an Allen wrench, turn the blade holder into the safe position and open the jaws to insert a blade.</li>
	<li>Tighten the jaws with an Allen wrench and turn the blade holder into the appropriate position for slicing.</li>
	<li>Place the sample and the platform onto the box and slide in front of the blade.</li>
	<li>Fill the box around the sample platform with 1x PBS until the sample is submerged.</li>
	<li>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.</li>
	<li>Confirm the appropriate settings and run the vibratome (Figure 2).</li>
	<li>As fresh slices are cut, remove the samples from the PBS and place them into a sterile Petri dish with 10 mL of 1x DMEM containing 1x antibiotic-antimycotic.</li>
	<li>When finished, retract the blade entirely and then use the Allen wrench to raise the blade into a safe position.</li>
	<li>Store the samples in 1x DMEM in an incubator at 37 &#176;C with viability preserved for up to 10 days (see viability experiment below).</li>
</ol>
4. Example vasoconstrictor experiment
<ol>
	<li>Place a vibratome slice on a microscope slide.</li>
	<li>Before placing it on the slide, use a cleaning wipe to get rid of the excess medium (PBS).</li>
	<li>Place the sample under phase-contrast microscopy and use 10-20x magnification to identify a vessel.</li>
	<li>Put 500 &#181;L of vasoconstrictors, such as 60 mM KCl or 1 &#181;M Endothelin-1 to completely submerge the slide.</li>
	<li>Observe and capture the video by recording for 30 s-60 s (Video 1).</li>
</ol>
5. Preparing the tissue for RNA or protein lysis on PCLS
<ol>
	<li>Before beginning the experiment, fill a small polystyrene foam box with 1 L of liquid nitrogen.</li>
	<li>Place two small microcentrifuge tubes (1.5 mL) in the container with liquid nitrogen before starting. 
	NOTE: Liquid nitrogen should cool outside of the tubes, however, not be inside. Be sure to handle liquid nitrogen with care and do not expose the skin to liquid nitrogen. Use forceps and gloves to place and remove tubes from the box.</li>
	<li>Place 4-5 fresh vibratome slices in a mortar bowl.</li>
	<li>Pour 5-10 mL of liquid N2 on top of the slices in the mortar bowl.</li>
	<li>Quickly use the pestle to grind the flash freeze slices into powder before liquid nitrogen evaporates. 
	NOTE: Tissue should condense into a fine powder. If not, add additional liquid nitrogen until achieved.</li>
	<li>Using a steel laboratory scooper (prechilled with liquid nitrogen), scoop the powder into the two chilled microcentrifuge tubes.</li>
	<li>Lyse the powder by adding 500 &#181;L of RNA extraction reagent to proceed with RNA isolation or 500 &#181;L of RIPA buffer (contains 1x protease inhibitors) to proceed with protein lysis.</li>
	<li>Store the samples at -80 &#176;C until additional RNA isolation, BCA measurement, and western blot.</li>
</ol>
6. Determining viability
<ol>
	<li>After vibratome preparation, place two lung slices into a 24-well culture plate with 450 &#181;L of DMEM media and 50 &#181;L of cell viability reagent (ensure that the tissue is submerged entirely).</li>
	<li>Place the samples back into 5% CO2 at 37 &#176;C incubator overnight with minimal exposure to light.</li>
	<li>In the morning, observe the solution for color change. The blue solution turns pink when the tissue is viable.</li>
</ol>
7. Preservation of cell labeling
<ol>
	<li>Treat a transgenic Cdh5-CreERT2 crossed with Ai14 tdTomato mouse with an IP injection of Tamoxifen (2 mg/day) for a total of 5 days (total dose 10 mg Tamoxifen) to induce tdTomato label Cre positive cells.</li>
	<li>One week after injection, prepare the lungs using the above steps 1 to 3.</li>
	<li>After preparation of the precision-cut lung slice, place the tissue onto a microscope slide and place a coverslip on top of the tissue.</li>
	<li>Use a confocal microscope to detect tdTomato staining (using excitation wavelength 555 nm).</li>
</ol>

<ol>
	<li>Sparrow, D., Weiss, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+physiology.">Respiratory physiology.</a> Annual Review of Gerontology &amp; Geriatrics. 6, 197-214 (1986).</li><li>Gerckens, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+3D+lung+tissue+cultures+(3D-LTCs)+for+disease+modeling.">Generation of human 3D lung tissue cultures (3D-LTCs) for disease modeling.</a> Journal of Visualized Experiments: JoVE. (144), e58437 (2019).</li><li>Li, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserving+airway+smooth+muscle+contraction+in+precision-cut+lung+slices.">Preserving airway smooth muscle contraction in precision-cut lung slices.</a> Scientific Reports. 10 (1), 6480 (2020).</li><li>Rosales Gerpe, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+precision-cut+lung+slices+as+an+ex+vivo+tool+for+evaluating+viruses+and+viral+vectors+for+gene+and+oncolytic+therapy.">Use of precision-cut lung slices as an ex vivo tool for evaluating viruses and viral vectors for gene and oncolytic therapy.</a> Molecular Therapy: Methods &amp; Clinical Development. 10, 245-256 (2018).</li><li>Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+lung+physiology+in+health+and+disease+with+lung+slices.">Exploring lung physiology in health and disease with lung slices.</a> Pulmonary Pharmacology &amp; Therapeutics. 24 (5), 452-465 (2011).</li><li>Liu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+lung+slices:+An+ex+vivo+model+for+the+evaluation+of+antiviral+and+anti-inflammatory+agents+against+influenza+viruses.">Mouse lung slices: An ex vivo model for the evaluation of antiviral and anti-inflammatory agents against influenza viruses.</a> Antiviral Research. 120, 101-111 (2015).</li><li>de Graaf, I. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+incubation+of+precision-cut+liver+and+intestinal+slices+for+application+in+drug+metabolism+and+toxicity+studies.">Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies.</a> Nature Protocols. 5 (9), 1540-1551 (2010).</li><li>Alsafadi, H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+and+approaches+for+three-dimensional+precision-cut+lung+slices.+Disease+modeling+and+drug+discovery.">Applications and approaches for three-dimensional precision-cut lung slices. Disease modeling and drug discovery.</a> American Journal of Respiratory Cell and Molecular Biology. 62 (6), 681-691 (2020).</li><li>Morin, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+cut+lung+slices+as+an+efficient+tool+for+in+vitro+lung+physio-pharmacotoxicology+studies.">Precision cut lung slices as an efficient tool for in vitro lung physio-pharmacotoxicology studies.</a> Xenobiotica. 43 (1), 63-72 (2013).</li><li>Springer, J., Fischer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substance+P-induced+pulmonary+vascular+remodelling+in+precision+cut+lung+slices.">Substance P-induced pulmonary vascular remodelling in precision cut lung slices.</a> The European Respiratory Journal. 22 (4), 596-601 (2003).</li><li>Suleiman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Argon+reduces+the+pulmonary+vascular+tone+in+rats+and+humans+by+GABA-receptor+activation.">Argon reduces the pulmonary vascular tone in rats and humans by GABA-receptor activation.</a> Scientific Reports. 9 (1), 1902 (2019).</li><li>Rieg, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+agents+affect+the+tone+of+pulmonary+arteries+and+veins+in+precision-cut+lung+slices.">Cardiovascular agents affect the tone of pulmonary arteries and veins in precision-cut lung slices.</a> PLoS One. 6 (12), 29698 (2011).</li><li>Perez, J. F., Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+frequency+of+calcium+oscillations+induced+by+5-HT,+ACH,+and+KCl+determine+the+contraction+of+smooth+muscle+cells+of+intrapulmonary+bronchioles.">The frequency of calcium oscillations induced by 5-HT, ACH, and KCl determine the contraction of smooth muscle cells of intrapulmonary bronchioles.</a> The Journal of General Physiology. 125 (6), 535-553 (2005).</li><li>Deng, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulation+of+5-hydroxytryptamine+receptor+signaling+in+coronary+arteries+after+organ+culture.">Upregulation of 5-hydroxytryptamine receptor signaling in coronary arteries after organ culture.</a> PLoS One. 9 (9), 107128 (2014).</li><li>Sandker, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adventitial+dissection:+A+simple+and+effective+way+to+reduce+radial+artery+spasm+in+coronary+bypass+surgery.">Adventitial dissection: A simple and effective way to reduce radial artery spasm in coronary bypass surgery.</a> Interactive Cardiovascular and Thoracic Surgery. 17 (5), 784-789 (2013).</li><li>Naik, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-induced+smooth+muscle+cell+depolarization+in+pulmonary+arteries+from+control+and+chronically+hypoxic+rats+does+not+cause+myogenic+vasoconstriction.">Pressure-induced smooth muscle cell depolarization in pulmonary arteries from control and chronically hypoxic rats does not cause myogenic vasoconstriction.</a> Journal of Applied Physiology. 98 (3), 1119-1124 (2005).</li><li>Lopez-Lopez, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+induces+pulmonary+artery+endothelial+dysfunction+by+NADPH+oxidase+induction.">Diabetes induces pulmonary artery endothelial dysfunction by NADPH oxidase induction.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 295 (5), 727-732 (2008).</li><li>Gonzalez-Tajuelo, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+pulmonary+hypertension+associated+with+systemic+sclerosis+in+P-selectin+glycoprotein+Ligand+1-deficient+mice.">Spontaneous pulmonary hypertension associated with systemic sclerosis in P-selectin glycoprotein Ligand 1-deficient mice.</a> Arthritis &amp; Rheumatology. 72 (3), 477-487 (2020).</li><li>Bai, Y., Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+Ca2++sensitivity+of+airway+smooth+muscle+cells+in+murine+lung+slices.">Modulation of the Ca2+ sensitivity of airway smooth muscle cells in murine lung slices.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 291 (2), 208-221 (2006).</li><li>Nishiyama, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+function+and+endothelin-1:+tipping+the+balance+between+vasodilation+and+vasoconstriction.">Vascular function and endothelin-1: tipping the balance between vasodilation and vasoconstriction.</a> Journal of Applied Physiology. 122 (2), 354-360 (2017).</li><li>Schneider, M. P., Inscho, E. W., Pollock, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuated+vasoconstrictor+responses+to+endothelin+in+afferent+arterioles+during+a+high-salt+diet.">Attenuated vasoconstrictor responses to endothelin in afferent arterioles during a high-salt diet.</a> American Journal of Physiology. Renal Physiology. 292 (4), 1208-1214 (2007).</li><li>Inscho, E. W., Imig, J. D., Cook, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Afferent+and+efferent+arteriolar+vasoconstriction+to+angiotensin+II+and+norepinephrine+involves+release+of+Ca2++from+intracellular+stores.">Afferent and efferent arteriolar vasoconstriction to angiotensin II and norepinephrine involves release of Ca2+ from intracellular stores.</a> Hypertension. 29, 222-227 (1997).</li><li>Vecchione, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+from+angiotensin+II-mediated+vasculotoxic+and+hypertensive+response+in+mice+lacking+PI3Kgamma.">Protection from angiotensin II-mediated vasculotoxic and hypertensive response in mice lacking PI3Kgamma.</a> The Journal of Experimental Medicine. 201 (8), 1217-1228 (2005).</li></ol>

The authors have no conflicts of interest to disclose.

In this manuscript, an enhanced method to produce high-resolution images of murine lung tissue that preserves the vascular structure and optimizes experimental flexibility is described, specifically using the application of PCLS to obtain microslices of lung tissue that can be viewed in three dimensions with preserved contractility of the vasculature. Using the viability reagent, the protocol demonstrates that carefully prepared and preserved slices can retain viability for more than a week. Preserved viability of the mi...

Advances in the preparation and imaging of lung tissue that preserves cellular components without sacrificing anatomical structure provide a detailed understanding of pulmonary diseases. The ability to identify proteins, RNA, and other biological compounds while maintaining physiological structure offers vital information on the spatial arrangement of cells that can broaden the understanding of the pathophysiology in numerous pulmonary diseases. These detailed images can lead to a better understanding of pulmonary vascular diseases, such as pulmonary artery hypertension, when applied to animal models, potentially leading to improved therapeutic strategies.
Despite advances in technology, obtaining high-quality images of murine lung tissue remains a challenge. The respiratory cycle is driven by a negative intrathoracic pressure generated during inhalation1. When traditionally obtaining biopsies and preparing lung samples for imaging, the negative pressure gradient is lost resulting in the collapse of the airway and vasculature, which no longer represents itself in its present state. To achieve realistic images reflective of current conditions, the pulmonary airways must be reinflated, and the vasculature perfused, changing the dynamic lung into a static fixture. The application of these distinct techniques allows preservation of structural integrity, pulmonary vasculature, and cellular components, including immune cells such as macrophages, allowing lung tissue to be viewed as close to its physiological state as possible.
Precision cut lung slicing (PCLS) is an ideal tool for studying the anatomy and physiology of pulmonary vasculature2. PCLS provides detailed imaging of the lung tissue in three dimensions while preserving structural and cellular components. PCLS has been used in animal and human models to allow for live, high-resolution images of cellular functions in three dimensions, making it an ideal tool to study potential therapeutic targets, measure small airway contraction and study the pathophysiology of chronic lung diseases such as COPD, ILD, and lung cancer3. Using similar techniques, the exposure of PCLS samples to vasoconstrictors can preserve lung structure and vessel contractility, replicating in vitro conditions. Along with preserving contractility, prepared samples can undergo additional analysis such as RNA sequencing, Western blot, and flow cytometry when prepared correctly. Finally, reporter color labeled cells marked with tdTomato fluorescence after lung harvest can preserve labeling after preparing microslices, making it ideal for cell tracking studies. The integration of these techniques provides a sophisticated model preserving the spatial arrangement of cells and vessel contractility that can lead to a more detailed understanding of the signaling cascades and potential therapeutic options in pulmonary vasculature disease.
In this manuscript, PCLS murine lung tissue is exposed to vasoconstrictors, demonstrating preserved structural integrity and vessel contractility. The study demonstrates that the tissue prepared and handled appropriately can remain viable for 10 days. The study also demonstrates the preservation of cells with endogenous fluorescence (tdTomato), allowing samples to provide high-resolution images of the pulmonary vasculature and architecture. Finally, ways to handle and prepare tissue slices for RNA measurement and Western blot to investigate underlying mechanisms have been described.

<table><tbody><tr><td>0.5cc of fractionated heparin in syringe</td><td>BD</td><td>100 USP units per mL</td><td></td></tr><tr><td>1X PBS</td><td>Corning</td><td>&amp;nbsp;21-040-CM</td><td></td></tr><tr><td>20 1/2 inch gauge blunt end needle for trachea cannulation</td><td>Cml Supply</td><td>90120050D</td><td></td></tr><tr><td>30cc syringe</td><td>BD</td><td>309650</td><td></td></tr><tr><td>Anti Anti solution</td><td>Gibco</td><td>15240096</td><td></td></tr><tr><td>Automated vibrating blade microtome</td><td>Leica</td><td>VT1200S</td><td></td></tr><tr><td>Cell Viability Reagent (alamarBlue)</td><td>Thermofisher</td><td>DAL1025</td><td></td></tr><tr><td>Confocal</td><td>Zeiss</td><td>880</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium and GLutaMAX, supplemented with 10% FBS, 1% Pen/Strep</td><td>Gibco</td><td>10569-010</td><td></td></tr><tr><td>Endothelin-1</td><td>Sigma</td><td>E7764</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>7447-40-7</td><td></td></tr><tr><td>Mortar and Pestle</td><td>Amazon</td><td></td><td></td></tr><tr><td>RIPA lysis and extraction buffer</td><td>Thermoscientific</td><td>89900</td><td></td></tr><tr><td>Surgical suture 6/0</td><td>FST</td><td>18020-60</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Invitrogen, Thermofisher</td><td>15596026</td><td></td></tr><tr><td>UltraPure Low Melting Point Agarose</td><td>Invitrogen</td><td>16520050</td><td></td></tr><tr><td>Vibratome</td><td>Leica Biosystems</td><td>VT1200 S</td><td></td></tr><tr><td>Winged blood collection set (Butterfly needle) 25-30G</td><td>BD</td><td>25-30G</td><td></td></tr></tbody></table>

precision cut lung slices as an efficient tool for ex vivo pulmonary vessel structure and contractility studies

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, the preservation of pulmonary vessels that truly represents physiological conditions still presents challenges. In addition, the delicate configuration of murine lungs result in technical challenges preparing samples for high-quality images that preserve both cellular composition and architecture. Similarly, cellular contractility assays can be performed to study the potential of cells to respond to vasoconstrictors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these technical issues, the precision-cut lung slice (PCLS) method can be applied as an efficient alternative to visualize lung tissue in three dimensions without regional bias and serve as a live surrogate contractility model for up to 10 days. Tissue prepared using PCLS has preserved structure and spatial orientation, making it ideal to study disease processes ex vivo. The location of endogenous tdTomato-labeled cells in PCLS harvested from an inducible tdTomato reporter murine model can be successfully visualized by confocal microscopy. After exposure to vasoconstrictors, PCLS demonstrates the preservation of both vessel contractility and lung structure, which can be captured by a time-lapse module. In combination with the other procedures, such as western blot and RNA analysis, PCLS can 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.

When added to cells or tissue, the viability reagent is modified by the reducing environment of viable tissue and turns pink/red, becoming highly fluorescent. The representative color changes detected from day 0-1 and day 9-10 are demonstrated in Figure 3. As noted, the solution started blue and turned pink overnight, demonstrating viability. Color change typically occurs within 1-4 h; however, a longer time may be necessary. To assay for viability, a plate reader was used to determine the a...

Watch this Scientific Journal Video about Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies at JoVE.com

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

Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

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

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, ...

precision-cut-lung-slices-as-an-efficient-tool-for-ex-vivo-pulmonary

Research

JoVE Journal

Medicine

5.7K Views.  Boston Children's Hospital. Precisie gesneden longplakken methode zal de structuur en ruimtelijke oriëntaties in drie dimensies behouden zonder regionale vertekening en kan dienen als een live surrogaatmodel voor vaten en luchtwegcontractiliteitsmetingen. In combinatie met time-lapse beeldvorming, Western blot en RNA-analyse dragen nauwkeurig gesneden longplakken bij aan het uitgebreide begrip van signaleringscascades die ten grondslag liggen aan een breed scala aan aandoeningen en leiden tot een beter begrip van de pa...

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

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 &#181;m in diameter3.
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 &#181;m inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 &#181;m which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 &#181;m which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2 and the response fades after 30-40 min at hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological phenomenon by which at alveolar hypoxia lung perfusion is matched to ventilation. The major vascular segment contributing to HPV is the intra-acinar artery. Here, we describe our protocol for the analysis of HPV of murine pulmonary vessels with diameters of 20-100 &mu;m.

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Immunology and Infection

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 &#181;m-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells.

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...

Bioengineering

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue&#39;s stiffness, which is crucial for subsequent slicing. 500 &#181;m thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Biology

Innovative Lung Imaging Using Agarose-Embedded Vibratome Sections

Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural support for sectioning, pieces of lung tissue are commonly embedded in paraffin or OCT compound and cut with a microtome or cryostat, respectively. A more recent technique, known as precision-cut lung slices, adds structural support to fresh lung tissue through agarose infiltration and provides a platform to maintain primary lung tissue in culture. However, due to epitope masking and tissue distortion, none of these techniques adequately lend themselves to the development of reproducible advanced light imaging readouts that would be compatible across multiple antibodies and species. 
To this end, we have developed a tissue-processing pipeline, which utilizes agarose embedding of fixed lung tissue, coupled to automated vibratome sectioning. This facilitated the generation of lung sections from 200 &#181;m to 70 &#181;m thick, in mouse, pig, and human lungs, which require no antigen retrieval, and represent the least "processed" version of the native isolated tissue. Using these slices, we reveal a multiplex imaging readout capable of generating high-resolution images whose spatial protein expression can be used to quantify and better understand the mechanisms underlying lung injury and regeneration.

Author Spotlight: Studying Spatial Protein Expression Using Agarose Embedded Lung Tissue Sections 

We have developed a tissue-processing technique utilizing a vibratome and agarose-embedded lung tissue to generate lung sections, thereby permitting the acquisition of high-resolution images of lung architecture. We employed immunofluorescence staining to observe spatial protein expression using specific lung structural markers.

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural ...

Utilizing the Precision-Cut Lung Slice to Study the Contractile Regulation of Airway and Intrapulmonary Arterial Smooth Muscle

Smooth muscle cells (SMC) mediate the contraction of the airway and the intrapulmonary artery to modify airflow resistance and pulmonary circulation, respectively, hence playing a critical role in the homeostasis of the pulmonary system. Deregulation of SMC contractility contributes to several pulmonary diseases, including asthma and pulmonary hypertension. However, due to limited tissue access and a lack of culture systems to maintain in vivo SMC phenotypes, molecular mechanisms underlying the deregulated SMC contractility in these diseases remain fully identified. The precision-cut lung slice (PCLS) offers an ex vivo model that circumvents these technical difficulties. As a live, thin lung tissue section, the PCLS retains SMC in natural surroundings and allows in situ tracking of SMC contraction and intracellular Ca2+ signaling that regulates SMC contractility. Here, a detailed mouse PCLS preparation protocol is provided, which preserves intact airways and intrapulmonary arteries. This protocol involves two essential steps before subjecting the lung lobe to slicing: inflating the airway with low-melting-point agarose through the trachea and infilling pulmonary vessels with gelatin through the right ventricle. The PCLS prepared using this protocol can be used for bioassays to evaluate Ca2+-mediated contractile regulation of SMC in both the airway and the intrapulmonary arterial compartments. When applied to mouse models of respiratory diseases, this protocol enables the functional investigation of SMC, thereby providing insight into the underlying mechanism of SMC contractility deregulation in diseases.

The present protocol describes preparing and utilizing mouse precision-cut lung slices to assess the airway and intrapulmonary arterial smooth muscle contractility in a nearly in vivo milieu.

Smooth muscle cells (SMC) mediate the contraction of the airway and the intrapulmonary artery to modify airflow resistance and pulmonary circulation, ...

Lung-tumor Tissue Explant Culture: A Procedure to Generate and Culture Precision-cut Lung Tumor Slices

Source: Nagaraj, A. S., et al. <a href="https://www.jove.com/v/58569/establishment-analysis-tumor-slice-explants-as-prerequisite-for">Establishment and Analysis of Tumor Slice Explants as a Prerequisite for Diagnostic Testing.</a>&#160;J. Vis. Exp.&#160;(2018)
Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the multicellular interactions of native tissue. This video describes the culturing of murine lung tumor-derived tissue slices using a rotating incubation unit, a system that enables intermittent exposure of tissues to oxygen and nutrients. The protocol describes the methodology to generate and culture precision-cut lung tumor slices for drug response studies.

Source: Nagaraj, A. S., et al. Establishment and Analysis of Tumor Slice Explants as a Prerequisite for Diagnostic Testing.&#160;J. Vis. Exp.&#160;(2018)
...

JoVE Encyclopedia of Experiments

Cancer Research

Lung Cancer

Ex vivo study

Agarose-Based Preparation of Human Precision-Cut Lung Slices

Source: Neuhaus, V. et al., <a href="https://app.jove.com/v/57042">Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices.</a> J. Vis. Exp. (2018)
In this video, we describe a procedure to inflate isolated human lungs with agarose and prepare human precision-cut lung slices. The prepared ex vivo lung slices can be used for further immunologic experiments.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. General Preparation of Human PCLS ...

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

Samenvatting

Meer video's verkennen