TITLE: Rodent Liver 2-Step Collagenase Perfusion and Digestion using VitaCyte
AUTHORS: sai.chung, manmeet.sekhon, Xue-Zhong Ma, Justin Manuel, Michael L Cheng, Sonya MacParland, Ian McGilvray

[DESCRIPTION]
To obtain a viable single cell suspensions of total liver homogenate (TLH) that can be used for downstream experiments, such as in vitro experiments and more biologically representative transcriptomics with greater success, a slow in situ 2-step collagenase rodent liver perfusion and digestion is developed in both mice and rat. The process of dissociation can accelerate cell death if done harshly. Thus, an optimized gentle method will yield more viable cells, which this protocol has been inspired by the human caudate 2-step perfusion and digestion within the lab. VitaCyte neutral protease and collagenase is used because of its high purity of the enzymes. 

[BEFORE_START]
Please read the protocol first to work efficiently and prepare reagents as well materials to reduce the time required. Also, account for the number of materials and reagents required, to the number of animals being perfused and digested. Furthermore, work efficiently after the liver is extracted, to decrease the time before downstream experiments/analyzes to prevent any further cell death.

[GUIDELINES]
All biological samples and animals should be handled and processed according to the institutes guidelines, eg. University Health Network (UHN).

[STEPS]
SECTION: Buffers and Solutions
1. Solution 1 – EGTA, Ca2+Mg2+ Free
MOUSE: Add 100µL of 50mM EGTA into 50mL of Ca2+Mg2+free HBSS. 
RAT: Add 500µL of 50mM EGTA into 250mL of Ca2+Mg2+free HBSS.
 

SECTION: Buffers and Solutions
2. Dehydrate
Add 68.6µL of 1M CaCl solution into 50mL of Ca2+Mg2+free HBSS. 
SECTION: Buffers and Solutions
3. Solution 2 – 2,000 NPU/mL collagenase and 500 NPU/mL protease with Ca2+Mg2+
MOUSE: Add 68.6µL of 1M CaCl solution and 500µL of HEPES into 49.43mL of Ca2+Mg2+free HBSS. 
RAT: Add 343µL of 1M CaCl solution and 2.5mL of HEPES into 247.16mL of Ca2+Mg2+free HBSS. 
 

SECTION: Buffers and Solutions
3.1. MOUSE:
Add 200µL of collagenase and 50µL of protease during the time dehydrate solution is being pumped. The enzymes will be thawed in their respective aliquots before dehydration and kept in the fridge to preserve enzymatic activity until added.

RAT:
Add 1250µL of collagenase and 275µL of protease during the time dehydrate solution is being pumped. The enzymes will be thawed in their respective aliquots before dehydration and kept in the fridge to preserve enzymatic activity until added.
SECTION: Buffers and Solutions
4. 0.2M NaOH
Add 1mL of 10N of NaOH into 49mL of MiliQ water. Always add base to water slowly in a
fume hood.
SECTION: Buffers and Solutions
5. Mincing Media (keep cold)
When opening a new 500mL DMEM, extract 5mL of DMEM and add 5mL of
penicillin/streptomycin to a final concentration of 1%. 
SECTION: Before Perfusion
6. Organize the surgical platform and BSC with all the materials and pre-labeled tubes prior to liver perfusion to obtain total liver homogenate (TLH) to be processed quickly.
SECTION: Before Perfusion
7. Place prepared solution 1, dehydrate and solution 2 (enzyme free) into an incubator
at 37 °C  with loosened caps for 40 minutes or directly into 42 °C water bath with caps
on firmly. 

SECTION: Before Perfusion
8. Prepare 2 tubes of 50mL each of dH2O, 70% EtOH and 1x PBS.
SECTION: Before Perfusion
9. Clean surgical platform. 
SECTION: Before Perfusion
10. Ensure anesthesia machine has enough oxygen and isofluorane for your procedure.
SECTION: Before Perfusion
11. Place a paper towel into anesthesia chamber.
SECTION: Before Perfusion
12. Fill a 1mL syringe with a 27G with heparin (stock, 1U/µL).
SECTION: Before Perfusion
13. Right before perfusion, clean the perfusion machine. 
SECTION: Anesthetizing the rodent
15. Re-tighten the caps of the buffers and transfer from the incubator to the water bath if not done so in previous section.
SECTION: Before Perfusion
14. Quickly, run 50mL of NaOH solution, followed by 50mL of milli-Q water or sterile dH2O then 50mL of ethanol and then finally 50mL of PBS. 
SECTION: Anesthetizing the rodent
16. Fill a 1mL syringe with warm Solution 1. Use the p200 to fill the dead space of the catheter.

MOUSE: 24G catheter
RAT: 20G catheter
SECTION: Anesthetizing the rodent
17. Turn on the oxygen flow rate at 3.5 L/min and isofluorane at 4%.
SECTION: Anesthetizing the rodent
18. Place the rodent into the anesthesia chamber.
SECTION: Anesthetizing the rodent
19. Observe breathing rate. 
SECTION: Anesthetizing the rodent
20. Once breathing has slowed, switch valves to direct flow to the tube and waste valves.
SECTION: Anesthetizing the rodent
21. Remove the rodent and tape it to the surgical platform with its snout in the anesthesia tube/hose.
 

SECTION: Anesthetizing the rodent
22. Decrease oxygen to 2 L/min and isofluorane to 2-2.5%.
SECTION: Anesthetizing the rodent
23. Observe the rodent's behavior. If its heartbeat/breathing is erratic (a strong intermittent pulsation is observed on its chest cavity), reduce the levels of isofluorane. Observe until slow steady breathing is achieved. 
SECTION: Cannulation
24. Shave the abdominal region of the mouse. 
SECTION: Cannulation
25. Wipe the surgical region with ethanol-soaked wipes.
SECTION: Cannulation
27. Wait 1 minute. Ensure breathing is still occurring normally.
SECTION: Cannulation
26. Inject the rodent peritoneally with:
MOUSE: 100U of heparin (100µL of stock heparin)
RAT:  400U of heparin (400µL of stock heparin diluted with 600µL PBS)
SECTION: Cannulation
28. Using the medium sharp forceps, pinch the skin in the middle of the lowest abdomen. Create a small incision using the sharp scissors. 
SECTION: Cannulation
29. Push the scissor through the separate the abdominal skin from the abdominal muscle. Create an incision running through the anterior-posterior axis of the rodent's abdomen. 
SECTION: Cannulation
31. Repeat the steps 29 and 30 for the abdominal muscles of the rodent.
SECTION: Cannulation
30. Make lateral incisions/cuts of the abdomen/skin beneath the thoracic cage. 
 

SECTION: Cannulation
32. Hold the flap away from the surgery area by clamping it with the large forceps and securing it by inserting the finger holes/rings through the plastic screws on the upper right and left of the operation platform.
 

SECTION: Cannulation
33. Put surgical microscope over the surgical area to view the image. 
SECTION: Cannulation
34. Push the liver up and the guts away from the inferior vena cava (IVC) and portal vein (PV).
SECTION: Cannulation
35. Observe the status of the IVC. If the IVC is deflated, then proceed the latter steps using the PV first.  

If both IVC and PV are defalted, then the success of the perfusion is low regardless. If this is true, suggest not to proceed and start again with a new rodent if concerned about cell yield.

SECTION: Cannulation
36. Observe the tissue surrounding the IVC. If the IVC is not clear and there are many layers of connective peritoneal tissue, then peel off these layers carefully with 2 forceps to not the tear the vessel until you reveal the IVC. An exposed IVC should be a deep clear burgundy red with no opaqueness.
 

SECTION: Cannulation
37. Cannulate the IVC below the kidneys with the (mouse: 24G, rat: 20G) catheter/syringe filled with solution 1 from earlier. Secure wth micro-vasculature clamp.
 



 

SECTION: Cannulation
39. After step 39, the PV should be slightly under pressure and visibly prominent. Remove any obstructing fat layers away from the PV delicately without tearing the vessel.
SECTION: Perfusion
43. After 1 minute and liver looks pale* instead of burgundy red, disconnect from PV catheter and connect to the IVC catheter (same flow rate for mouse but 16mL/min for rat). The liquid running out of the top of the PV catheter should be clear.
 

SECTION: Perfusion
42. Start the perfusion pump flow of solution 1 from the IVC. Observe for bubbles, if it is clear - connect the perfusion tube to the PV cannula and let it run at 1-2mL/min for mouse (or 12mL/min for rat) for 1 minute whilst shaking the lobes of the liver to ensure proper perfusion.   
 

 

SECTION: Perfusion
44. Run 2-3 more minutes while shaking the liver until satisfactorily clear.
 

SECTION: Perfusion
44.1. During this time, thaw 1 aliquot of VitaCyte collagenase and protease. Once thawed, place vials in the  4°C fridge or keep on ice.
SECTION: Perfusion
45. Pause the pump and switch to dehydrate solution. 

MOUSE: Run for 2 minutes. 
RAT: Run for the entire 50mL dehydrate solution.
SECTION: Perfusion
45.1. During this time, add collagenase and protease into solution 2 and invert the sealed tube several times.
SECTION: Digestion
47. Run solution 2 while shaking the liver, then disconnect after 15-22 minutes (or 3/4 of the solution 2 running time) switch to PV perfusion for the remainder of the digestion. 
 


 


SECTION: Digestion
46. Pause the perfusion run and switch to solution 2. 
SECTION: Liver Extraction
48. Carefully remove by lifting the liver up via the PV and remove the liver by removing the tissue connecting to the stomach and small intestines, as well as the posterior abdominal wall, kidneys and the IVC.
SECTION: Mincing the liver for TLH collection
52. Place the perfused and digested rodent liver in a petri dish containing (mouse: 10mL, rat: 50mL) of cold mincing media on an ice pack. 
 

SECTION: Mincing the liver for TLH collection
50. For rats, place a 500µm cell strainer on top of a 250mL sterile collection vessel on ice for each rat being perfused.

Skip to the next step for mice.

SECTION: Liver Extraction
49. Place the liver into a petri dish with (mouse: 10mL, rat: 50mL) of cold mincing media. 
SECTION: Buffers and Solutions
1.1. MOUSE:
Add 80U of heparin (80µL of stock heparin) into solution 1 and invert the tube before running it through the pump.

RAT:
Add 400U of heparin (4mL of stock heparin) into solution 1 and invert the tube before running it through the pump.

RAT:
Add 400U of heparin (400µL of stock heparin) into solution 1 and invert the tube before running it through the pump.
SECTION: Mincing the liver for TLH collection
53. Flip the liver and swirl it around and flip it back gently.
SECTION: Mincing the liver for TLH collection
54. Make multiple vertical thin cuts. While doing this, occasionally swirl the liver. 
SECTION: Mincing the liver for TLH collection
55. Carefully, place the top of the petri dish under the dish on one end to cause the liver contents to be on the other side the dish (tilted down towards the operator). Chop the liver contents in this soup multiple times.
SECTION: Mincing the liver for TLH collection
56. Pipette up and down multiple times to dissociate any remaining tissue with a 25mL serological pipette. 
SECTION: Mincing the liver for TLH collection
58. Transfer the TLH into the (mouse: 50mL tubes, rat: 250mL collection vessels) with 100µm cell strainer (1 vessel for each animal).
 

SECTION: Mincing the liver for TLH collection
59. Transfer the TLH into the (mouse: 50mL tubes, rat: 250mL collection vessels) with 100µm cell strainer into the appropriate vessels with 70µm cell strainer (1 vessel for each animal).
 

 

SECTION: Mincing the liver for TLH collection
60.     
Perform trypan blue on TLH by adding 20µL of cells into 180µL of PBS and take 10µL of 1:10 diluted cells with 10µL of trypan blue. Load 10µL of the trypan blue with cells (1:20) into hemocytometer to count cells of interest (ex. hepatocytes or non-parenchymal cells).
 

 

SECTION: Anesthetizing the rodent
23.1. Pinch its hind paws for a pain withdrawal reflex. If no paw withdrawal reflex is observed, proceed to operation.
SECTION: Cannulation
24.1. Remove of excess hairs with Kim wipes and any residual with tape.
SECTION: Cannulation
27.1. During this time, wipe the surgical platform and surrounding area with Virox wipes for any residual fur.
SECTION: Cannulation
34.1. Locate the PV and IVC. 
 

SECTION: Cannulation
34.2. Use the locking clamp to secure the guts away from the surgical area if in the way, not absolutely necessary.
SECTION: Troubleshooting
61. If the IVC and/or PV cannot be located or is teared, then clamp the superior vena cava (SVC) or the
side vasculatures. 
SECTION: Troubleshooting
62. If the lobes are not inflated with solution 1 running, then increase the flow rate to ensure the edges are being flushed and perfused. But if the lobes are too inflated during the perfusion, then decrease the flow rate, thus decreasing the pressure and preventing cell damage. 
SECTION: Cannulation
38. To increase the subsequent PV cannulation success, the slow removal of the cannulae needle (not the cannulae catheter), a small amount of solution 1 at 0.5-1mL/min is pumped.


 


 

SECTION: Cannulation
38.1. Stop the pump. Disconnect the tubing from the needle and connect it to the cannulae. 
SECTION: Buffers and Solutions
5.1. MOUSE:
Add 5mL of FBS into 45mL of DMEM with 1% penicillin/streptomycin. 

RAT:
Add 15mL of FBS into 135mL of DMEM with 1% penicillin/streptomycin. Or make three 50mL tubes of mincing media if a container that can hold a large volume is not available.
 

SECTION: Buffers and Solutions
5.2. Filter the media using 50mL syringe and 0.22µm syringe filter before use. 

 

SECTION: Cannulation
40. Insert a (mouse: 24G, rat: 20G) cannulae into the PV, and secure with micro-vasculature clamp.
SECTION: Cannulation
37.1. Ensure there are no bubbles at the tip of the catheter or inside the syringe chamber. 
 



SECTION: Cannulation
40.1. Remove the cannulae needle. 

 


SECTION: Cannulation
39.1. Use the small tweezer to pinch the tissue around the PV, and lift/secure the PV.  
SECTION: Cannulation
41. Ensure angle of the catheter is fixed and secure parallel to the blood vessel to not disrupt flow of the solution running.
 

SECTION: Mincing the liver for TLH collection
51. Place a (mouse: 50mL tube, rat: 250mL collection vessel) with a 100µm cell strainer and a (mouse: 50mL tube, rat: 250mL collection vessel)  with 70µm cell strainers in the ice box for each rodent being perfused. 
 

SECTION: Mincing the liver for TLH collection
57. Skip this step for mice.

For rats, transfer the TLH into the 250mL vessel with 500µm cell strainer (1 vessel for each rat). Proceed to next step 59.