TITLE: Production of High-Purity Cell Suspensions from Human Precision Cut Lung Slices for Single-Cell RNA Sequencing 
AUTHORS: Michael Peasley, William_Bender, Alexis Owen, Heidie Huyck, Gautam Bandyopadhyay, Jeffrey Malik, Ravi Misra, John Ashton, Edward E. Walsh, Gloria S Pryhuber, tom mariani, Christopher S. Anderson

[DESCRIPTION]
Background:
In vitro models reduce the risk to humans caused by medical research but are
often limited to a single or few cell types. Precision Cut Lung Slices (PCLS), which
model the human lung tissue, contain major cell types in situ allowing the preservation
of intra cell type interactions that occurs during both homeostasis and disease
states. Here we demonstrate a procedure to produce high-quality single-cell RNA
sequencing data from cultured human Precision Cut Lung Slices.
Methods: Cryogenically
frozen PCLS slices were thawed and cultured on Transwell membranes. Slices were
put into contact with media, but not submerged. Slices were fed every other day
for 8 days and put into single cell suspension by enzymatic dissociation.
Mechanical separation using cell strainers and density gradient centrifugation
was used to deplete residual agarose. Single cell suspensions were captured and
sequenced.
Results: Single-cell
suspensions from PCLS slices yielded greater than 80% viable cells. Mechanical
and density gradient centrifugation reduced both the amount and the size of agarose
in the single-cell suspensions. A total of 34,069 cells were captured for
single-cell RNA sequencing, 97.4% of Barcodes were valid, and 97.4% of Reads
were Mapped to Genome. 16 cell types, including all major cell types in the
lung (endothelial, epithelial, mesenchymal, and immune), were identified.
Conclusion:
The described method produced single-cell suspensions from Precision Cut Lung
Slices that resulted in high-quality single-cell RNA sequencing data of all
major cell types.

[STEPS]
SECTION: PPE:
1. Lab coat, gloves and face shield should be worn at all times. All consumables (e.g. tips) should be disinfected in 10% bleach after use. Surfaces should be cleaned prior and after use with Virex followed by 70% ethanol. Experimenter should not open door to hall while working with tissue. No one is allowed into the tissue culture room while the tissue is being actively worked on. All work should be done at a BSL2 level. 
SECTION: PCLS-TW Media Prep
2. Combine the following to the top chamber of a bottle-top vacuum filter:
500 mL DMEM high glucose (4.5g/L) + L-Glutamine 
50 mL Heat-inactivated FBS
5 mL of 100x Non-Essential Amino Acids 
5 mL Sodium Pyruvate 100nM
7.5 mL 1M HEPES Buffer
5 mL of 10,000u/mL Penicillin/Streptavidin 
500 µL (125ug) Amphotericin B
800 µL (40ug) Gentamicin

Using vacuum, filter media into bottom chamber. Aliquot and store at 4 °C until needed.

SECTION: Tissue Growth (Day 1 &amp; 2)
3. Prep:
Warm water bath containing autoclaved diH2O to 37°C
SECTION: Tissue Growth (Day 1 &amp; 2)
4. Day One:
Obtain vial(s) of PCLS and place vial into warm water bath. Making sure not to submerge the cap and gently swirl vial until only small ice crystals remain. Do not thaw completely. 
SECTION: Tissue Growth (Day 1 &amp; 2)
5. In biosafety cabinet, decant the vial swiftly over a transwell membrane of a 6-well transwell membrane letting the slice wash out. If the slice does not easily fall out, rinse the vial with PBS, repeat as needed. 
If necessary, sterile flat head forceps may be used to gently seize the slice and place atop the transwell membrane. Be sure to uncurl the slice so it lies flat.
SECTION: Tissue Growth (Day 1 &amp; 2)
6. Gently dispense 500 µL of DPBS with Ca2+/Mg2+ onto the slice. Gently swirl plate and then remove DPBS, making sure not the disturb the slice. Repeat.
SECTION: Tissue Growth (Day 1 &amp; 2)
7. Dispense 800 µL of PLCS-TW media into the bottom chamber of the transwell. Media should come into contact with PCLS, but not submerge PCLS.
Label each well of transwell plate accordingly. 
SECTION: Tissue Growth (Day 1 &amp; 2)
8. To ensure wells do not dry out during incubation, dispense 800 µL of PLCS-TW media to any unused wells.
SECTION: Tissue Growth (Day 1 &amp; 2)
9. Place plate in incubator at 37°C with 5% CO2.
SECTION: Tissue Growth (Day 1 &amp; 2)
10. Day Two: 
Remove transwell plate from the incubator and move to biosafety cabinet. 
SECTION: Tissue Growth (Day 1 &amp; 2)
12. Dispense 800 µL of fresh PCLS-TW media into the bottom chamber of each well. 

SECTION: Single Cell Dissociation
14. Prep: 
- Set incubator to 37°C
- Turn on centrifuge and set the temperature to 4°C 
- Place the following into the biosafety cabinet:
Pasteurs
p1000
1000uL tips 
Tube holder
Forceps
Chlorox spray bottle 
15mL tube holder
Bleach beaker 
Dissociation cocktail 
SECTION: Single Cell Dissociation
15. Dissociation Cocktail:
1. Digestion Buffer (may be made ahead, store at 4°C):
a) Divalent cation free DPBS (1 L) 
+ 10mM HEPES-NaOH (pH 7.4) (use 10mL of 1M stock)
+ 150mM NaCl (8.77g)
+ 5mM KCl (0.37g)
+ 1mM MgCl2 (0.1g)
+ 1.8mM CaCl2 (0.2g)
2.  Make dissociation cocktail then add the following enzymes: 
a) Collagenase Type A from Clostridium; 2mg/ml final (powder) (0.15units/mg and 0.30 units/mL). Stored as lyophilized dry powder at 2-8°C, dry enyzme is stable for 6-12 months. 
b) Dispase II; 1mg/ml final (liquid), 1:5 dilution of total digestion cocktail volume (50 units/mL stock, 10 units/mL).
c) Elastase: Twice crystallized from Porcine Pancreas (0.5mg/mL final) (3 units/mg and 1.5 units/mL). Elastase in unstable at pH 3.5. Elastase product codes: ES and ESL have poor solubility at neutral pH and at concentrations greater than 0.25%. It is suggested that primary solutions be made in KCl or alkaline buffers and diluted into the reaction mixtures or media, compensating for ionic strength of pH changes. 
3. DNase-Deoxyribonuclease-1 from Bovine pancreas; 2mg/mL final (powder). 
a) 10mg/mL solution of DNase 1 in 0.15 M NaCl may lose <10% of its activity when stored for a week in aliquots at -20°C. The remains active for up to five hours at 60°C between pH 5 and 7, and loses activity in <10 minutes at 68°C. Activity of 1 mg/mL solution is lost at the rate of 6%/hour in acetate buffer (pH 5) or tris buffer (pH 7.2)  

For 10 mL of dissociation cocktail (in a 15 mL conical):

+  10 mL of Digestion Buffer
+ 20 mg Collagenase type A
+ 200 µL Dipase II (1 mg/ mL)
+ 5 mg Elastase
+ 20 mg DNase

SECTION: Single Cell Dissociation
16. Measure and dispense 500 µL of dissociation cocktail into appropriately labeled 5mL culture tubes, one tube per slice.
SECTION: Single Cell Dissociation
17. Place each PCLS into separate 5mL culture tubes containing dissociation cocktail.
Label the tubes accordingly.
SECTION: Single Cell Dissociation
18. Place tubes on orbital shaker located in a 37°C incubator.
SECTION: Single Cell Dissociation
19. Incubate and shake  .
SECTION: Single Cell Dissociation
20. Place the following items inside the biosafety cabinet: 
Tray full of wet ice
70uM cell strainers 
6 well place on ice 
DPBS with Ca2+/Mg2+ (DPBS++) on ice
Labeled 15mL tubes 
3mL syringes 
SECTION: Single Cell Dissociation
21. After the 40 minute incubation, add 500 µL DPBS++ to each tube and pour into cell strainer on top of a well. 
SECTION: Single Cell Dissociation
22. Rinse each set of tubes with an additional 500 µL of DPBS ++ to maximize cell recovery. 
SECTION: Single Cell Dissociation
23. Push tissue through the cell strainer with the plunger end of the syringe. Gently swirl in circular motions, repeat 5-10 times.
Note: Samples can be pooled at this time if needed.
SECTION: Single Cell Dissociation
24. Add 500 µL of DPBS++ and push remaining tissue through the cell strainer with the plunger end of the syringe.
Note: Large agarose pieces and fibrous tissue may remain.  

SECTION: Single Cell Dissociation
25. Collect strained cells using pipette and place in 15mL tube. 
SECTION: Single Cell Dissociation
26. Centrifuge 15mL tube 1000 x g, 10 min, 4 °C 

SECTION: Debris Removal (Performed on 0.5-1 g estimated pooled tissue)
27. Aspirate supernatant completely. 
SECTION: Debris Removal (Performed on 0.5-1 g estimated pooled tissue)
28. Resuspend cell suspension carefully with 6200 µL of cold buffer (DPBS++) and transfer cell suspension to a 15mL tube. Do not vortex. 

SECTION: Debris Removal (Performed on 0.5-1 g estimated pooled tissue)
29. Add 1800 µL of cold Debris Removal Solution. 

SECTION: Debris Removal (Performed on 0.5-1 g estimated pooled tissue)
30. Mix well by pipetting 10 times slowly up and down using a 2 mL pipette.
SECTION: Debris Removal (Performed on 0.5-1 g estimated pooled tissue)
31. Overlay very gently with 4 mL of cold buffer (DPBS++) making sure to tilt tube and pipette very slowly to ensure that the PBS/D-PBS phase overlays the cell suspension and phases are not mixed. 
SECTION: Debris Removal (Performed on 0.5-1 g estimated pooled tissue)
32. Centrifuge 3000 x g, 10 min, 4 °C with full acceleration and full brake. Three phases are formed. 
Note: The acceleration and brake can be reduced to increase separation of phases. 
SECTION: Debris Removal (Performed on 0.5-1 g estimated pooled tissue)
33. Vacuum aspirate the top two phases completely and discard (approximately 4mL).
SECTION: Debris Removal (Performed on 0.5-1 g estimated pooled tissue)
34. Add cold DPBS++ to a final volume of 15 mL

SECTION: Debris Removal (Performed on 0.5-1 g estimated pooled tissue)
35. Gently invert the tube three times to mix. Do not vortex. 
SECTION: Debris Removal (Performed on 0.5-1 g estimated pooled tissue)
36. Centrifuge 1000 x g, 10 min, 4 °C with full acceleration and full brakes.
SECTION: Debris Removal (Performed on 0.5-1 g estimated pooled tissue)
37. Aspirate supernatant completely making sure not to disturb pellet.
SECTION: Debris Removal (Performed on 0.5-1 g estimated pooled tissue)
38. Carefully resuspend cells by adding 175 µL of cold DPBS++ by pipetting slowly up and down. Do not vortex. Place on ice.
SECTION: Debris Removal (Performed on 0.5-1 g estimated pooled tissue)
39. Count the cells. 
SECTION: Tissue Growth (Day 1 &amp; 2)
13. Return transwell plate to incubator at 37°C with 5% CO2.
SECTION: Tissue Growth (Day 1 &amp; 2)
11. Aspirate the media from bottom chamber of each well and discard.