Rat hepatocyte isolation assay
Rat hepatocyte isolation assay
Although cells isolated from adult livers do not express all the properties of the liver parenchyma, there is no doubt that suitable cell lines can be cultured. To date, the desire to establish continuously proliferating cell lines has not been fully realized. However, it is possible to culture functional hepatocytes under appropriate conditions. Source: Animal Cell Culture: A Guide to Basic Techniques, Fifth Edition
Operation method
Scheme 23.6 Rat hepatocyte isolation assay
Principle
The liver was cannulated through the hepatic portal vein or a branch of the hepatic portal vein and lavaged with calcium-free buffer for 15 min, followed by enzyme solution for 15 min. Cells were collected and washed, and viable hepatocytes were counted.
Materials and Instruments
L-15 Leibovitz culture solution Ham F12 culture solution Cytosolic culture solution HMM SF Calcium-free HEPES buffer Collagenase solution 5% Sodium pentobarbital Heparin Move I. Materials For more product details, please visit Aladdin Scientific website.
Polyethylene tubing Disposable 20 G scalp IV needles Sutures l Syringes Water baths Bat pumps
1. Aseptic
(1) L-15 Leibovitz culture liquid
(2) Ham F12 culture medium or WilliamE culture medium: 80-100 ml, containing 0.2% bovine albumin (grade V, Sigma) and 1 ug/ml bovine insulin.
(3) hepatocyte minimalmediu (HMM): 67.5% EMEM and 22.5% 199 culture medium, or William E culture medium instead of these two mediums. Add 5ug/ml bovine insulin, lmg/ml BSA, 20 mmol/L sodium pyruvate, 100U/ml penicillin, 100 Mg/ml streptomycin and 20% FBS.
(4) HMM/SF: serum-free HMM, add 10-5mol/L hydrocortisone hemisuccinate
(5) Calcium-free HEPES buffer: containing 160.8 mmol/L NaCl, 3.15 mmol/L KCl, 0.7 mmol/L Na2 HPO4-12H2O, and 33 mmol/L HEPES, pH 7.65. Filtered through 0.22, microporous filters for sterilization, and stored at 4°C for 2 months.
(6) Collagenase Solution: Contain 0.025% collagenase (Grade I, Sigma; or Boche, 103578) and 0.075% CaCl2-2 H2O in calcium-free HEPES buffer pH 7.65. The buffer was prepared before use and filtered for sterilization.
(7) 5% sodium pentobarbital (Abbott)
(8) Heparin (Roche)
(9) Polyethylene tubing: inner diameter 3 mm, outer diameter 5 mm
(10) Disposable 20 G cephalic intravenous infusion needle (Dubernard Hospital Laboratory, Bordeaux, France).
(11) Suture for cannulation
(12) Graduated bottles and Petri dishes
(13) Surgical instruments: sharp scissors, straight scissors, curved scissors and surgical clips
(14) 2 secondary 1 ml syringes
2. non-sterile
(1) Timer
(2) Batting pump: 10～200r/min
(3) Water bath tank
Second, the operation steps
1. Preheat the HEPES Buffer Wash and Collagenase Solution in a water bath to 38-39°C, or 37°C for intrahepatic administration. Oxygenation is not required.
2. Set the flow rate of the screw rack at 30 ml/min.
3. Anesthetize rats (180-200 g ) by injecting sodium pentobarbital (150 ul per 100 g body weight) into the peritoneal cavity. Then, 10001U of heparin was injected via femoral vein.
4. The ventral surface of the rats was scrubbed with 70% ethanol.
5. The abdominal wall was incised and a ligature line was tied to the hepatic portal vein at approximately 5 mm from the liver. The hepatic portal vein is ligated after cannulation in the direction of the liver.
6. To avoid overpressurization, the hepatic vein is quickly clipped. Perfuse 500 ml of calcium-free HEPES buffer at a flow rate of 30 ml/min. Confirm whitening of the liver within a few seconds.
7. Perfuse 300 ml of collagenase solution at a flow rate of 15 ml/min. The liver becomes swollen.
8. Remove liver and rinse with HEPES buffer.
9. After stripping the Glisson's capsule, the cells were dispersed in 100 ml of L-15 Leibovitz culture medium.
10. Filter the cell suspension through two layers of gauze or nylon mesh with a pore size of 60-80um. The viable cells were allowed to settle for 20 min, usually at room temperature. Then, aspirate 60 ml of supernatant containing cell debris and dead cells.
11. Wash the cells 3 times by slow centrifugation (50 g, 40 s) to remove collagenase, damaged cells and non-substantial cells.
12. Suspend the isolated hepatocytes with HMM.
13. After 2-3 h, the live cells will attach to the wall and begin to stretch. Aspirate off the supernatant containing dead cells.
14. After the cells have attached to the wall, replace the HMM with HMM/SF containing FBS. thereafter, change the culture medium daily.