TITLE: Protocol for Primary Mouse Hepatocyte Isolation -- University of Minnesota TMCs
AUTHORS: Laura Niedernhofer, David Bernlohr, Linshan Shang

[DESCRIPTION]
STAR Protocol for Primary Mouse Hepatocyte Isolation -     

Protocol used for isolating primary hepatocytes to be used in scRNAseq and bulk RNA seq. 

[BEFORE_START]
BEFORE YOU BEGIN
 
1. Prepare cell culture plates; 12-well, 6-well, 10 cm, and 15 cm plates are suitable for hepatocyte plating.
 

2. Cover the bottom of the plates/wells with 0.01% rat-tail collagen solution.
3. Incubate for 4–16 h at 37 °C under sterile conditions (e.g., in a humidified CO2 incubator).
4. Wash with PBS, aspirate PBS.
 

STEP-BY-STEP METHOD DETAILS

This protocol is aimed at isolating hepatocytes from mouse liver. Following anesthesia, the vena cava is cannulated and the liver is perfused to chelate calcium and wash out blood. Then, collagenase is perfused to the liver in order to dissociate extracellular matrix. Finally, the liver is dissected and hepatocytes are purified by density-based separation. This protocol presents several advances over similar protocols (Berry and Friend, 1969; Casciano, 2000; Klaunig et al., 1981; Li et al., 2010; Renton et al., 1978; Seglen, 1976; Severgnini et al., 2012). The main improvements of this protocol are better reproducibility, shortened duration, reduced technical challenge, increased yield and higher viability. These are achieved by several steps we altered or optimized. For example: (a) We found that retrograde perfusion through the vena cava permits easier cannulation as opposed to portal vein cannulation. (b) Periodical clamping of the portal vein provides a visible checkpoint for proper perfusion and greatly facilitates efficient washing and digestion. (c) The type of collagenase used (Liberase) shows quicker digestion and better reproducibility compared to other collagenases. (d) Percoll-based density separation results in a population of purified hepatocytes of high viability. Some of these protocol improvements were already implemented in our previous publications (Goldstein et al., 2017a; Goldstein et al., 2017b) where we isolated hepatocytes for experiments
demanding a high yield of cells (such as chromatin immunoprecipitation sequencing – ChIP-seq).
  


[STEPS]
SECTION: Pump Preparation and Mouse Anesthesia
1. 
 
Warm water bath to 42 °C.
SECTION: Pump Preparation and Mouse Anesthesia
2.  Place perfusion buffer in the water bath.
SECTION: Pump Preparation and Mouse Anesthesia
3. Prepare the peristaltic pump:
SECTION: Pump Preparation and Mouse Anesthesia
3.1. Run 70% ethanol through the tubing.
SECTION: Pump Preparation and Mouse Anesthesia
3.2. Run air through the tubing for 30–60 s.
SECTION: Pump Preparation and Mouse Anesthesia
3.3. Wipe the end of the tubing with a paper towel.
SECTION: Pump Preparation and Mouse Anesthesia
3.4. Connect 27-gauge needle to the outlet end of the tubing using a luer lock (Figure 1).
 
SECTION: Pump Preparation and Mouse Anesthesia
4. Prime the tubing with warm perfusion buffer (pump speed 3 mL/min).
 


SECTION: Pump Preparation and Mouse Anesthesia
5. Prepare two 50 mL tubes (labeled ‘‘DB-chilled’’ and ‘‘DB-Liberase’’), each with 10 mL digestion buffer. Keep the tube labeled ‘‘DB-chilled’’ on ice. Add 250 µL Liberase solution to the tube labeled ‘‘DB-Liberase’’ and warm it in the water bath.
SECTION: Pump Preparation and Mouse Anesthesia
6. Anesthetize mouse by intraperitoneal injection of anesthesia mix (3.75 µL/g body weight. Final concentration for ketamine = 112.5 mg/kg; for xylazine =22.5 mg/kg).
 

SECTION: Pump Preparation and Mouse Anesthesia
7.  Place mouse on the edge of the dissection tray and secure limbs using needles (Figure 2).
 
  
SECTION: Pump Preparation and Mouse Anesthesia
8. Wet the fur thoroughly with 70% ethanol.
SECTION: Pump Preparation and Mouse Anesthesia
9. Make a ‘‘U’’-shaped incision through the skin, secure the skin near the head using a needle (Figure 3).
  
SECTION: Pump Preparation and Mouse Anesthesia
10. Move the intestine to the right to reveal the portal vein and vena cava (Figure 4). Place a stable and heavy object adjacent to the mouse hind legs to support the tubing such that it is slightly higher than the mouse, lay the tubing and the needle on the object (Figure 1). The edge of the needle should rest on the vena cava in a flat angle (i.e., parallel to the vein, Figure 5).
 
    
SECTION: Cannulation and Perfusion
11. 
 
Turn on the pump and let the warm perfusion buffer reach the needle (the buffer within the
tubing has already cooled down to 20-25 °C).
 


SECTION: Cannulation and Perfusion
12. While buffer is running through the needle, insert the needle into the vena cava above the kidney (Figure 5).
 

SECTION: Cannulation and Perfusion
13. Immediately upon appearance of white spots and/or portal vein swelling (occurs 2–3 s after cannulation), cut the portal vein with scissors. The liver should clear of blood instantly; you’ll observe
plenty of blood rushing out of the portal vein and the liver will turn yellow-white within a few seconds.
SECTION: Cannulation and Perfusion
14. Clamp the portal vein with forceps for 7–10 s (Figures 6A and 6B). Make sure no fluid is passing
through.
 
 
SECTION: Cannulation and Perfusion
15. After 30 s perform a second clamp, make sure you observe liver swelling and relaxation. If the liver is completely washed of blood, move to the next section.

SECTION: Digestion
16. 
 
Stop the pump and quickly transfer the inlet tubing from perfusion buffer to the pre-warmed
‘‘DB-Liberase’’ tube. Quickly turn the pump back on.
 

SECTION: Digestion
17. Clamp the liver one more time, prior to digestion buffer arriving to the liver. Make sure the liver
swells and relaxes.
SECTION: Digestion
18. While digestion buffer is perfused into the liver, clamp the portal vein every minute but no more
than 3–4 times.
 

SECTION: Digestion
19. Remove the needle before air gets into the liver.
SECTION: Digestion
20. Dissect out the liver gently (it is now very flimsy and frail): using forceps, grab the central connective tissue between the lobes and slightly lift upwards, using it as an anchor point. Cut all the connections of the liver to other organs, remove the gall bladder, place the liver in the tube labeled ‘‘DB-chilled’’ and put it back on ice. 
 

SECTION: Digestion
21. Clean the pump with ethanol (see step 3).
SECTION: Hepatocyte Purification
22. 
 
Transfer liver and media to a 10 cm plate (not pre-coated with collagen).
SECTION: Hepatocyte Purification
23. Rupture liver sack with fine tip forceps in a few locations along the liver surface and gently release cells using a cell lifter.
 

SECTION: Hepatocyte Purification
24. Filter 5 mL of suspension through a   cell strainer into a 50 mL tube. Repeat with a new filter and a new tube for the remaining 5 mL.

SECTION: Hepatocyte Purification
25. Add another 10 mL of cold plating media to rinse plate, add 5 mL of it to each filter.

SECTION: Hepatocyte Purification
26. Spin at 50 x g, 2 min, 4 °C (Figure 7A).
 
  
SECTION: Hepatocyte Purification
27. While the samples are spinning, prepare fresh Percoll solution from pre-chilled ingredients.
 

SECTION: Hepatocyte Purification
28. Aspirate most supernatant, leave ∼1 mL and resuspend the cells by swirling the tube.

SECTION: Hepatocyte Purification
29. Add 10 mL plating media and resuspend by gentle swirling.

SECTION: Hepatocyte Purification
30. Add 10 mL Percoll solution and mix thoroughly by inverting the tube several times.

SECTION: Hepatocyte Purification
31. Spin at 200 x g, 10 min, 4 °C (Figure 7B).
 
  

SECTION: Hepatocyte Purification
32. Aspirate most supernatant, leave ∼1 mL and resuspend the cells by swirling the tube.
 


SECTION: Hepatocyte Purification
33. Aspirate supernatant, add 20 mL Williams E media

SECTION: Hepatocyte Purification
34. Suspension stored on ice and submitted for single cell capture using 10x Chromium platform