TITLE: Enzymatic liver dissociation (with liver perfusion kit)
AUTHORS: Dorien De Pooter, Ben De Clerck, Koen Dockx, Domenica De Santis, Sarah Sauviller, Pascale Dehertogh, Matthias Beyens, Isabelle Bergiers, Isabel Nájera, Ellen Van Gulck, Nádia Conceição-Neto, Wim Pierson

[DESCRIPTION]
This protocol details enzymatic liver dissociation. 

[STEPS]
SECTION: Reagent preparation
1. Storage

Lyophilized enzymes should be stored at 2 °C–8 °C. Buffer P (20x), Reagent C and Reagent E should be stored at Room temperature. Lyophilized enzymes should be reconstituted before the date indicated on their respective vials.
SECTION: Reagent preparation
2. Reconstitution buffer
SECTION: Reagent preparation
3.1. Enzyme D 

Prepare Enzyme D by reconstitution of the lyophilized powder in the vial with 3 mL reconstitution buffer. Invert the vial after closing the lid and wait 5 min–10 minto dissolve the powder. Do not pipette up and down to dissolve the enzyme! 
Prepare aliquots to avoid repeated freeze-thaw cycles. Store aliquots at -20 °C. 
 

SECTION: Reagent preparation
3.2. Enzyme R 

Prepare Enzyme R by reconstitution of the lyophilized powder in the vial with 3 mL reconstitution buffer. Invert the vial after closing the lid and wait 5 min–10 min to dissolve the powder. Do not pipette up and down to dissolve the enzyme! 
Prepare aliquots to avoid repeated freeze-thaw cycles. Store aliquots at -20 °C. 

 

SECTION: Reagent preparation
3. Enzyme reconstitution 
SECTION: Reagent preparation
3.3. Enzyme A 

Prepare Enzyme A by reconstitution of the lyophilized powder in the vial with 1 mL of reconstitution buffer. Do not vortex! 
Prepare aliquots to avoid repeated freeze-thaw-cycles. Store aliquots at -20 °C. 

 

SECTION: Procedure : PART I
9. A) General remarks:

A maximum of 1.2 g of liver tissue can be perfused in one GentleMACS Perfuser. 
SECTION: Procedure : PART I
9.1. The time from the dissection of the liver to the start of the perfusion should not exceed 10 min. Longer time spans will impact cell yield and viability. 
SECTION: Procedure : PART I
9.2. The adjuster of the lid of the GentleMACS Perfuser is already provided in the correct position (original position) for the perfusion of mouse liver. Do not turn the adjuster if mouse liver will be perfused! 
SECTION: Procedure : PART I
9.3. The position of the adjuster already altered by accident, it can be re-adjusted to the original position by turning it counterclockwise to the highest position and then slightly back until the “.5 mark” on the adjuster’s scale matches the arrowhead on the adjuster. 

 

SECTION: Procedure : PART II
11. A) Mouse liver perfusion: 
SECTION: Procedure : PART II
11.1. Carefully dissect the left lateral liver lobe. Rinse the lobe in a 100 mm tissue culture dish filled with 1× PBS.

 
  
SECTION: Procedure : PART II
11.3. Squeeze the clamp and attach it to the grid by pushing it down until the clamp touches the surface of the petri dish to fix the liver between grid and clamp. 

 

SECTION: Procedure : PART II
11.4. Remove the lid of the GentleMACS perfuser and connect it by pushing the grid holder through the clamp into the grid containing the tissue. 

 

SECTION: Procedure : PART II
11.5. Transfer the assembly of lid-clamp-grid back onto the base of the GentleMACS perfuser. 
SECTION: Procedure : PART II
11.6. Turn the lid slowly counterclockwise without pressure on the base until the lid drops to a lower position. Then, screw the thread of the lid clockwise without tilting to tightly close the GentleMACS Perfuser. 
SECTION: Procedure : PART II
11.7. Resume the program which is paused after priming to continue with the initial perfusion step. 

 

SECTION: Procedure : PART II
11.8. After the initial perfusion step, the program will pause to start the washing phase. This phase consists of 4 cycles of buffer exchange, and the program will pause after each cycle. 

 

SECTION: Procedure : PART II
11.9. During the pause, remove the used pre-digestion buffer manually with a VACUSAFE aspiration system and then add 8 mL of fresh pre-digestion buffer. Resume the program only after the buffer has been exchanged. 
SECTION: Procedure : PART II
11.10. After the last washing step, remove the pre-digestion buffer and add 8 mLequilibration buffer. Resume the program only after the buffer has been exchanged.
SECTION: Procedure : PART II
12. B) Enzymatic liver digest:
SECTION: Procedure : PART II
12.1. Prepare the enzyme digestion mix for 1 perfusion during the equilibration phase. 

 
 
SECTION: Procedure : PART II
12.3. After the enzymatic perfusion step, the program ends. Do not discard the used enzyme digestion mix.
SECTION: Procedure : PART II
13. C) Mechanical dissociation of digested liver:
SECTION: Procedure : PART II
13.1. Detach the heating unit and GentleMACS perfuser from the GentleMACS instrument. 
SECTION: Procedure : PART II
13.3. Pour the used enzyme digestion mix from the base into a GentleMACS C Tube and discard the base. 
SECTION: Procedure : PART II
13.4. Push up the clamp using tweezers and transfer the tissue from the grid into the C Tube containing the used enzyme digestion mix. Discard the lid with attached clamp-grid assembly. 
SECTION: Procedure : PART II
13.5. Tightly close the C Tube and attach it upside down onto a regular GentleMACS sleeve of the GentleMACS Dissociator. 

 

SECTION: Procedure : PART II
13.6. Run the GentleMACS program LIPK_HR_1.

 
 
SECTION: Procedure : PART II
13.7. Put a 15 mL tube on ice and place a MACS SmartStrainer (100 μm) on it. 

 

SECTION: Procedure : PART II
13.8. After termination of the program, detach the C Tube from the GentleMACS Dissociator. 
SECTION: Procedure : PART II
13.9. Open the tube and transfer the cell suspension onto the MACS SmartStrainer (100 μm). 
SECTION: Procedure : PART II
13.10. Wash the C Tube with 6 mLof liver wash buffer and transfer this onto the MACS SmartStrainer (100 μm). 

SECTION: Procedure : PART II
13.11. Discard the MACS SmartStrainer (100 μm) and close the 15 mL tube.
SECTION: Procedure : PART II
13.12. Gently invert the tube several times to ensure proper mixing of the liver wash buffer and the digested liver solution.
SECTION: Procedure : PART II
13.13. Keep the cells on ice until further processing.

SECTION: Reagent preparation
2.1. Prepare the reconstitution buffer by diluting 400 µL Buffer P (20×) with 7.6 mL sterile water.
 
 
 
SECTION: Reagent preparation
2.3. Buffer P (20×) is susceptible to bacterial contamination. Sterile aliquoting of Buffer P (20×) is recommended. 
Store buffer P (20×) at room temperature and prewarm the buffer at 37 °C for at least 45 min before first use. Shake the buffer thoroughly before use. 
SECTION: Reagent preparation
2.2. Add 16 µL of Reagent C and mix well.
 

SECTION: Reagent preparation
4. Perfusion buffers
For 62 mL Buffer P (1x):
 
 
Buffer P (1x) is used as the basis to prepare the perfusion buffers. 

 

SECTION: Reagent preparation
6. Equilibration buffer 

Transfer 18 mL of Buffer P (1x) to a new 50 mL tube and add 36 µL Reagent C. 
Take 9 mL of the prepared equilibration buffer and transfer to a separate tube. 
This equilibration buffer will be used to prepare the enzyme digestion mix.

 

SECTION: Reagent preparation
5. Pre-digestion buffer 

Transfer 44 mL of Buffer P (1x) to a new 50 mL tube.
SECTION: Reagent preparation
7. Enzyme digestion mix 

Prepare the enzyme digestion mix during the equilibration phase. Enzymes must be added freshly, shortly before the enzyme digestion mix is used. 
 
 
 
SECTION: Reagent preparation
8. Liver Wash Buffer 

Thaw a 50 mL aliquot of FCS and prepare a solution of 9% FCS (v/v) in DMEM (1x) + 4.5g/l D-glucose + L-glutamine. This buffer can be stored at 4 °C for 3 months. 
SECTION: Procedure : PART I
10. B) Preparation:

Ensure that the regular GentleMACS sleeves have been exchanged by the perfusion sleeves before attaching the GentleMACS perfuser.
SECTION: Procedure : PART I
10.1. Pre-heat water bath with beads at 37 °C. 
SECTION: Procedure : PART I
10.2. Pre-warm pre-digestion buffer and tubes of equilibration buffer at 37 °C. 
SECTION: Procedure : PART I
10.3. Attach the base of the GentleMACS perfuser with the lid onto the GentleMACS perfusion sleeve on the GentleMACS Octo Dissociator with heaters. Leave out the grid and the clamp.
 

SECTION: Procedure : PART I
10.4. Check the adjuster in the correct position for liver tissue perfusion.
SECTION: Procedure : PART I
10.5. Place a heating unit onto the GentleMACS perfuser.
SECTION: Procedure : PART I
10.7. Start the appropriate GentleMACS program for mouse 37C_m_LIPK_1.
SECTION: Procedure : PART I
10.6. Carefully transfer 8 mL of warm pre-digestion buffer into the GentleMACS perfuser by using one of the two luer lock connectors.
SECTION: Procedure : PART I
10.9. Place the grid into a 100 mm tissue culture dish.
SECTION: Procedure : PART I
10.8. After the priming step, the program will automatically pause and will be resumed later after the tissue sample is loaded.
SECTION: Procedure : PART II
11.2. Place the liver lobe in the center of the grid. 
SECTION: Procedure : PART II
12.2. After the equilibration step, remove the equilibration buffer and add 8 mL enzyme digestion mix. Resume program only after buffer has been exchanged.
SECTION: Procedure : PART II
13.2. Unscrew the lid and transfer the lid-clamp-grid assembly into a 60 mm tissue culture dish.