Adapted from Fraslin chicken hepatocyte isolation protocol

Warm to 37C: Perfusion buffer 1, Perfusion buffer 2, Supplemented media. Set heatblock to 42C to keep buffers slightly warmer during perfusion.
Work in situ on a freshly killed turkey (~5-7 week old Domestic turkey = ~2-4kg).
Insert catheter (~18ga X 2”) into the inferior mesenteric artery and suture in place. Snip vena cava between liver and heart to allow outflow.
Add 0.25-0.5mL Heparin (~875U/kg = 0.5mL Heparin (4400U/mL) for a 2.2kg bird) to a 50mL aliquot of Perfusion buffer 1. Perfuse (17mL/min) to start blanching while preventing clotting.
Perfuse (25ml/min) with Perfusion buffer 1 until blanched (~50-250mL). Gently “massage” red areas to help clear blood clots and achieve more even blanching.
Perfuse with Perfusion buffer 2 until digested (~250mL or when the liver stays dimpled when poked with a blunt tool). During perfusion put pressure on vena cava for a few seconds to inflate liver and assist in cell dissociation. Repeat every 2-3 minutes.
Excise the liver and move to the sterile hood for remaining steps.
Add 50-100mL Perfusion buffer 1 and release hepatocytes by breaking the glissons capsule and gently probing with a cell scraper.
Filter through cheesecloth to separate the undigested tissue. Pour ~50-100mL Perfusion buffer 1 over the undigested liver in the cheesecloth to maximize cell yield. Discard undigested tissue with cheesecloth.
Into 50mL falcon tubes, filter with nylon-mesh filters of pore sizes 100 (or 70um), and 40um to eliminate cell aggregates (turkey hepatocyctes are ~18uM—3 times smaller than mammalian hepatocytes).
Pellet hepatocytes by centrifugation (3 minutes @ 200g). Discard supernatant.
Gently resuspend cells in enough Supplemented media to make 13.25mL per percoll tube. In 50mL falcon tubes, place 13.25mL suspended cells and 20mL Diluted percoll (45%) to make a final concentration of 27% percoll. (Equation below for reference)
```
[Final percoll] = [starting percoll] * Volume of percoll)
               ---------------------------------------
                             Total volume
```
Pellet hepatocytes by centrifugation (5 minutes @ 100g). Pellet should contain red blood cells and a small amount of cells.
- Discard top ~ 5mL (Should be the lightest “garbage”)
- Collect as much supernatant as possible without mixing red blood cells back out of the pellet. Place supernatant in a new 50mL falcon tube and measure volume with a serological pipette.
- Discard the pellet (more red blood cells than hepatocytes).

Add media to the supernatant to reduce percoll from 27% to 21% according to the equation below:

Supp. media needed = 0.286 * Volume of supernatant)

(General form of the equation for reference):

Supp. media needed = (([starting percoll] - [desired percoll]) * Volume of supernatant)
              --------------------------------------------------------------
                                   [desired percoll]

Pellet hepatocytes by centrifugation (5 minutes @ 100g). Discard supernatant.
Wash away remaining percoll by adding 20-30mL Supplemented media and spinning 3 minutes @ 200g. Discard supernatant. Repeat wash at least once, but spin 1 minute @ 200g.
Gently resuspend and adjust volume of hepatocytes to ~5-10mL with Supplemented media. Measure volume with pipette to get accurate volume for concentration calculations.
Count cells and determine viability with hemocytometer and trypan blue. Calculate and record viable cell concentration.
Plate 125000 cells/well with 100uL Supplemented medium in 96-well plates. Incubate at 37C.
Optionally Cryogenically preserve excess cells.
Warm to 37C: Supplemented media, Definitive media.
After 2 to 3 h remove media from plated cells, gently wash cells once with Supplemented media, and add 200uL of Definitive media. Incubate.
Change media every other day.

RECIPES

5X Perfusion buffer (HBSS + HEPES)

136mM Sodium Chloride, 25mM HEPES, 5.5mM D-Glucose, 5.4mM Potassium Chloride, 4.2mM Sodium Bicarbonate, 0.9mM Magnesium Chloride, 0.5mM Magnesium Sulphate, 0.5mM EDTA, 0.45mM Potassium Phosphate, 0.35mM Sodium Phosphate, pH 7.4

40g Sodium Chloride
29.8g HEPES
5.0g D-Glucose
2.0g Potassium Chloride
1.8g Sodium Bicarbonate
915mg Magnesium Chloride hexahydrate
730mg EDTA
510mg Magnesium Sulphate heptahydrate
470mg Sodium Phosphate dibasic
305mg Potassium Phosphate monobasic
Add water to 950mL
Raise pH to 7.4 with NaOH.
Add water to 1L.

Perfusion buffer 1

1X HBSS + HEPES pH 7.4

160mL 5X Perfusion buffer
640mL water
Adjust pH to 7.4
Filter-sterilize.
Makes enough for:
- 50ml for Buffer1 + Heparin to stop clotting and start blanching
- 250mL for blanching (excess can also be used for breaking up the liver)
- 200mL for breaking up the liver
- 300mL for making Perfusion buffer 2

Perfusion buffer 2

1X HBSS + HEPES, 1.3mM Calcium Chloride, ~5U/mL collagenase type 4, pH 7.4

43mg Calcium Chloride in 300mL Perfusion buffer 1
Mix to dissolve completely.
Adjust pH to 7.4
Filter-sterilize.
Add 5mg collagenase type 4 (~305U/mg) just before use.

Supplemented media

100U/mL penicillin/streptomycin, 10ug/mL insulin

500 mL William’s E media
5mL penicillin/streptomycin
5mg insulin

Definitive media

1ug/mL glucagon, 100ug/mL transferrin, 0.4ug/mL dexamethasone

23.25mL Supplemented media
150uL glucagon (stock = 200ug/mL)
6mL transferrin (stock = 500ug/mL)
600uL dexamethasone (stock = 20ug/mL)
Optionally add 5%v/v FBS

Diluted Percoll

To make Stock Isotonic Percoll (SIP):

90mL stock Percoll (1.130g/mL density)
10mL 1.5M NaCl
Do this in the hood to keep the stock bottle of percoll sterile

To make Diluted Percoll (45%):

100mL SIP
100mL 1X HBSS + HEPES
Adjust pH to 7.4 with 30% HCl (it should take ~250uL).
Note- Only use exactly 30% HCL for this! If you use a stronger or weaker solution the final percoll% will be different. Even small changes to the percoll% will have large effects on the size and cleanliness of the cell pellet.