TITLE: Human-Derived Precision-Cut Lung Slices (hPCLS): Agarose Filling, Coring and Slicing Protocol
AUTHORS: Ricardo Pineda, John Sembrat, Carter Kessler, Nayra Cardenes, Melanie Königshoff

[DESCRIPTION]
 Human Precision-Cut Lung Slices (hPCLS) are uniform tissue slices generated from human lungs. These slices contain all resident lung cells and their interactions, including alveolar macrophages and dendritic cells, present in the tissue at the time of slicing, representing the natural complexity of the lung’s 3-dimensional architecture and organ function physiology.
 
hPCLS has become one of the most relevant ex vivo organotypic models for studying the pathophysiology of human lungs. Thanks to specialized tissue slicers, a single lung can provide hundreds of slices for both control and experimental conditions. Additionally, hPCLS can be kept in culture for up to 4 weeks, making possible to mimic the onset and progression of lung diseases.  
 
hPCLS has proven valuable in multiple applications such as RNA and protein analysis, biochemistry, metabolism, toxicology, and chronic and acute lung diseases. They have also been used for highly specialized downstream applications such as transcriptomics, proteomics and high-resolution live microscopy. 
 
The following protocol is a guide on how to generate PCLS from human lungs.

[BEFORE_START]
Set a water or bead bath at 42°C

[GUIDELINES]
All steps involving human tissue must be conducted under BSL2 safety guidelines.


[STEPS]
SECTION: Core Slicing
24. 

 

 

Materials:
• Agarose 2.0% in DMEM/F12 with antibiotics and fungizone warmed up and kept at 40-42°C
• Double edge razor blades cut in half lengthwise and cleaned with ETOH to remove protective wax and oils.
• Cyanoacrylate Glue (we like Loctite, super glue, gel control ).
• Bucket with ice 
• DMEM/F12 with 1% penstrep, 2.5 ug/ml amphotericin (optional), and 1.0% FBS 
• Fine painting brush (natural fibers, NOT SYNTHETIC)
• Two 100 mm Petri dishes
• Forceps
• Several 3 ml sterile plastic transfer pipettes
• Clean Kimwipes
• Silicon grease

SECTION: Lung Filling and Coring
9. 
 

It is recommended to take pictures of the lung tissue in different planes before agarose inflation for future records. Include a ruler for size reference.
SECTION: DMEM/F12 Preparation for Agarose or Complete media
2. If inflating lungs continue to steps on preparing Agarose (LMPA). If treating or storing the PCLS, continue to  next steps for complete medium (DMEM/F12).

 

SECTION: DMEM/F12 Preparation for Agarose or Complete media
3. Weigh 15g of LMPA for a final concentration of 2.5% LMPA in 600 ml of DMEM/F12.
  

SECTION: DMEM/F12 Preparation for Agarose or Complete media
4. Add the LMPA to 400 ml of DMEM/F12 media in a clean, sterile, wide-mouth bottle (we recommend the 500 ml Pyrex round media storage bottles), and mix. Let the agarose hydrate for about 5 minutes. A magnetic bar can be included in the bottle.
 

SECTION: DMEM/F12 Preparation for Agarose or Complete media
5. Microwave the media/LMPA mix on high for about 30 seconds and then mix. Repeat the heating and mixing occasionally until the agarose starts to dissolve (this can be done with a magnetic stirrer). DO NOT BOIL. When the agarose is completely dissolved and the media is transparent, let cool to approximately 50-55°C.
 

SECTION: DMEM/F12 Preparation for Agarose or Complete media
6. Add 6.0 ml of FBS (final concentration 1%), 12 ml of Pen/Strep 10x (final concentration 2X), and 9 ml of Amphotericin B stock solution (final concentration 1.5X, optional ). Mix well.
    

SECTION: DMEM/F12 Preparation for Agarose or Complete media
8. Keep this solution at 42°C in a water or bead bath until use.
SECTION: DMEM/F12 Preparation for Agarose or Complete media
7. Complete the volume to 600 ml with DMEM/F12 media at room temperature. Keep stirring. Warm it up a little if necessary to keep the agarose from gelling.
SECTION: DMEM/F12 Preparation for Agarose or Complete media
1.3. Adjust the pH with NaOH 1N to 7.2.
SECTION: DMEM/F12 Preparation for Agarose or Complete media
1.2. Add 1.2 g of sodium bicarbonate. Stir and bubble with carbogen (a gas mixture of 30% CO2 and 70% oxygen) until pH is about 6.8-7.0 (The solution will turn red at this point). Then, stop bubbling.
SECTION: DMEM/F12 Preparation for Agarose or Complete media
1.1. Dissolve the content of one DMEM/F12 pouch in 700 ml of dd-water (the solution will be yellow).
SECTION: DMEM/F12 Preparation for Agarose or Complete media
1. Due to the large amounts of media required, we recommend the use of reconstituted powder DMEM/F12. 
  

SECTION: DMEM/F12 Preparation
3. Make up the volume to 1000 ml in a volumetric flask with distilled water.
SECTION: DMEM/F12 Preparation
4. Sterile filter using a polyethersulfone (PES) 0.2 μm membrane into a clean, sterile bottle.
SECTION: DMEM/F12 Preparation
5. Add:
10 ml of Pen/Strep 10x for a final concentration of 1% Pen/Strep 1x.   
10 ml from a 250µg/ml stock solution of Amphotericin B for a final concentration of  2.5 µg/ml.   
Fetal Bovine Serum (FBS) at desired concentration if necessary for subsequent purposes.
SECTION: DMEM/F12 Preparation
6. Label bottles and store at 4 °C when not in use.
SECTION: Lung Filling and Coring
22. 
From different areas of the remaining thick tissue slices, cut a few small pieces of about 2-3 mm3 and distribute them into a 6-well plate. Add fresh DMEM/F12 media with 1% strep, 1x Amphotericin, 1% FBS, and incubate overnight at 37 °C in a quarantine incubator.


SECTION: Lung Filling and Coring
13. Use hemostats to clamp shut all arteries, exposed blood vessels, and any areas where the pleura has been cut or compromised.  Fill a 60ml syringe with warm agarose and attach it to the end of a cannula or flexible tubing, insert it into the main airway following its natural track being careful not to use excessive force or forcing the cannula in.
Using constant pressure and speed, inject the agarose solution into the lung.

 

SECTION: Lung Filling and Coring
10. If the lung has not been flushed or has too much blood or mucus in the airways, it will be necessary to flush it with saline and antibiotics before agarose inflation.
Fill a 60 ml syringe with sterile saline containing 2x Pen/Strep and 1.5x Amphotericin B (optional but recommended). Gently push the saline through airways and blood vessels to force blood and mucus out. Gently squish the saline out of the lung by compressing the tissue. Do not apply excessive force. Repeat the process until most of the blood and mucus have been cleared out. For the mid and lower left lobes, about 1 L of saline should suffice.
SECTION: Lung Filling and Coring
11. After flushing, rinse the tissue in a clean bucket twice with sterile saline and antibiotics.
SECTION: Lung Filling and Coring
15. Once one region of the lobe if full, relocate the inflating cannula to a different airway and continue filling with warm agarose until full inflation. Do not overfill.
Complete agarose filling will ensure structure preservation and will facilitate coring and slicing.
SECTION: Lung Filling and Coring
14. While injecting the agarose solution, check for leaks at the pleura or through blood vessels/airways.  Use hemostats to clamp shut any leaks. If the agarose solution start leaking out through the same airway it was injected, clamp it closed with a hemostat keeping the injecting cannula tightly fixed in place inside the airway.
SECTION: Lung Filling and Coring
12. Find the main bronchus and examine the opening to determine how many airways branch from it. This will guide the positioning of the cannula through the different airways to fill the lung.
SECTION: Lung Filling and Coring
17. After 45-60 minutes transfer tissue to a cutting board and remove all clamps.
SECTION: Lung Filling and Coring
16. After filling the lung lobe, clamp shut all airways and blood vessels that may leak. Place the tissue into a clean, new zip-lock plastic bag. Close it. Transfer the bag to an ice bucket and ensure complete coverage with ice for 45-60 minutes for the agarose to be completely solidified. 
Be careful not to allow ice or ice water to leak into the plastic bag.
SECTION: Lung Filling and Coring
19. Take pictures of the lung slices for future records.
SECTION: Lung Filling and Coring
18. Orient the tissue according to your experimental plan and slice the lobe into thick slices of about 2-2.5cm thick with a long trimming blade.
SECTION: Lung Filling and Coring
21. Collect the cores in a container with DMEM/F12 supplemented with 1% Pen/Strep, 1X of Amphotericin B (optional) and 1% FBS. Keep at 4°C until slicing. (Check Step-case 2 for Complete medium recipe).

 

SECTION: Lung Filling and Coring
20. With a tissue coring tool, punch out cylindrical cores from the areas of interest.
SECTION: Lung Filling and Coring
23. Check for possible contamination the next day.
If contamination is present, discard all cores.

25. From the Compresstome, take the white Teflon plunger out of the metal specimen tube and apply a light coat of silicone grease to the first half of it. Reinsert the piston into the metal tube. It should have a tight seal.
47. Once slicing is done, transfer the PCLS to a 24-well plate. One PCLS per well, with complete DMEM/F12 (see Step-case 2 for recipe).
46. If some agarose remains attached to the PCLS, hold the PCLS down by the agarose using a pair of forceps and gently “pull away” the tissue from the agarose matrix with the brush.
45. Transfer the PCLS into a petri dish kept on ice containing DMEM/F12 with antibiotics and 1.0% FBS.
44. Slicing should be continuous and shouldn't present any major problems.

 

43. The first slice should contain mostly agarose and if so, a little piece of tissue. This piece can be discarded.
42. Press the “continuous” key down and press “start”.
41. Press the “fast forward” button again to push the agarose without tissue out of the sample holder. About 1 mm.
40. Check the parameters for slicing PCLS: Speed 4.0, Oscillation 4.0, and slice thickness at 300 um.
39. Attach the blade holder to the vibratory arm and secure it firmly with the hex screw.
38. Press the “fast forward” button to move the metal guide forward until contacting the base of the Teflon plunger.
37. Fill the chamber with DMEM/F12 up to cover the sample holder.
36. 
Secure the slicing chamber with the safety screw.

 

35. Check that the metal guide from the Compresstome control panel is completely retracted into the control panel, otherwise the sample holder piston won’t fit in place.
34. Insert the specimen tube into the holder of the slicing chamber making sure not to push the agarose with the tissue out. It should have a tight, waterproof seal.
33. 

 
Once the agar in the canister is set and cool, take the specimen tube out of the ice, dry the metal surface, and apply a light coat of silicone grease around the center of the canister.
32. Attach one-half of the double-sided blade in the blade holder by applying a light coat of cyanoacrylate glue to the holder IF NECESSARY (newer Compresstome models have a magnetic blade holder). Center the blade and let the glue cure.
31. 

 
In the meantime, prepare the Compresstome: Put a light coat of silicone grease inside the specimen tube holder of the slicing chamber.
30. Make a big enough hole in the ice to put the specimen tube with the tissue in it to cool the agarose. Wait for the agarose to solidify.

 

 

29. With a plastic transfer pipette fill the specimen tube with enough warm agarose to cover up the tissue.

 

28. Pull the piston down into the specimen tube until the whole core is inside it.

 

27. Apply a light coat or a drop of the cyanoacrylate glue gel to the center top of the Teflon plunger keeping the metal specimen tube upright. Gently set the tissue core. Wait a few seconds until the glue is set.
26. Grab one lung core and remove any liquid excess with a new, clean Kimwipe. Do not overdry the tissue.