Anne Kienhuis | Jennifer Willet
1 lamb liver within 48 hours of slaughter
1 cooler w/ ice
1 cutting board
4 syringes – w/ terumo cannula
1 plastic tray
1 plastic stand w/ holes
1 filter + 100 mesh
10 ml pipettes
25 ml pipettes
6 well tissue culture tray
HBSS 10% solution w/ sterile water
DMEM + Glutamax + Collagenase (Add 250 mg Collagenase to 500 mL DMEM)
DMEM + Glutamax + Penstrep +10% FBS
DMEM + Glutamax + Penstrep
1) Put Liver on ice immediately after purchase, and transport to laboratory
2) Sterilize all equipment with 70% ethanol
3) Immediately (before the blood has much time to clot) slice a small piece of lobe off with a single clean cut.
4) Place liver on top of perforated stand – on top of a beaker - within a plastic tray.
5) Using a syringe and tc – perfuse exposed veins with PBS until color is drained from liver.
6) Choose three prominent vein entries into the liver (test their prominence by injecting HBSS solution into veins.
7) place three stationary syringes + tc into these openings, and crazy glue into place
8) Seal entire cut opening of liver lobe with the application of crazy glue
9) Simultaneously flush each opening with 100ml of HBSS solution SLOWLY.
10) Take a new beaker.
11) Simultaneously flush each opening with 100ml of DMEM w/ Collagenase solution SLOWLY – ensuring that all excess is captured by the beaker below.
12) Repeat three times
13) Remove plugs, and crazy glue carefully with tweezers
14) Take a moment to clean out the beaker with a towel and 70% ethanol (take care that ethanol is evaporated before use!)
15) Make several cuts into the liver.
16) Pour DMEM + FBS over cuts in liver – capturing the solution in your beaker. Do this several times. Eventually, stirring the liver gently in the beaker creating a cell suspension solution.
17) Filter into a clean beaker twice, once with the filter alone, and the second time with the 100 mesh nestled within the filter.
18) Pour final solution back into the DMEM + FBS 50ml vial.
19) Centrifuge for 5 min at 75g.
20) Remove excess DMEM with a pipette gun, and take off top layer of red blood cells and dead cells.
21) Add 10 ml of DMEM + FBS, pipetteing up and down stirring cells.
22) (it is good at this point to repeat steps 19 – to 21.
23) Place solution evenly in 6 well TC tray
24) Rotate tray in a figure 8 pattern evenly distributing the cells over the tray, and incubate at 37 C w/ CO2 5%.