Liver Perfusion - Hamsters Liver Perfusion and Hepatocytes isolation (Mar/11/2005 )
I am learning to perform liver perfusion in Hamsters. Surgery and perfusion are suucessfull, but at the end my cell viability is 0.00%
I perform the following protocol:
1. Adult Syrian-Golden male hamsters (140 to 160 g) are anesthetized with Isoflurane/O2 .
2. The abdomen is dissected and the abdominal aorta exposed. Fat is separated from the abdominal aorta with wet gauze.
3. Suture the abdominal aorta below the kidney in two positions and the thoracic aorta between the liver and the heart right underneath the lungs without tighten the suture. In the last process it is better to avoid cutting the diaphragm (Fig.1).
4. Insert the catheter (23G) below the first suture, extract the needle inside and replace it with the luer fitter connected to the butterfly needle and the pump.
5. Tighten the 3 sutures and close them with three knots.
6. Cut the portal vein to allow the exit of the perfusate.
7. [Bubble O2 in the media at 37oC through a 29G needle connected to a tube and the O2 cylinder] Skip this step for now.
8. Perfuse the liver with ~50 mL of warm (37¢XC) Liver Perfusion Medium [enriched in O2] through the abdominal aorta (legated below the kidneys and above the liver) at a rate of ~3.6 mL/minute with the perfusate exiting through the portal vein. The liver should change color and the perfusion can be stopped when the liquid flowing through the portal vein is free of blood.
9. This is followed by a digestion with ~25-30 ml of Liver Digest Medium at a rate of ~2.6 mL/minute. This results in blanching, softening and dissociation of liver tissue and provides complete digestion of the liver in 10-12 minutes. (The perfusion should be stopped when the liver tissue become soft and cracks after touching it with forcepts).
10. Aseptically remove the liver and transfer it to a sterile 100 mm dish containing 15ml of cold Hepatocytes Wash Media (keep on ice).
11. Remove bladder and connective tissue.
12. Mince cells finely with scissors.
13. Wash cells by replacing the Hepatocytes Wash Media with fresh one (15ml) two times.
14. After the last washing, filter cells suspension through a sterile 100 microm Cell Strainer into a 50ml sterile conical tube placed on ice
15. Adjust the volume to 45ml with Hepatocytes Wash Media
16. Centrifuge cells suspension 5 min at 50-60 g at 4oC
17. Remove the supernatant, adjust the volume to 25ml adding Hepatocytes Wash Media to the sedimented cells.
18. Centrifuge cells suspension 5 min at 50-60 g at 4oC
19. Remove the supernatant; adjust the volume to 10-20 ml with Williams E Medium containing 5% FBS and 10 microg/ml insulin. The volume of medium used depends on the size of the cells pellet
20. incubate cells suspension in a humidified atmosphere of 5% CO2 in air at 37C for 30 min
21. Count the cells with 0.4% Trypan Blue (1:1)
Any suggestion is welcome!
What we found that it was of some help for improving the health of the isolated hepatocytes (from mouse in my case) was to do a perfusion with wash buffer BEFORE the digestion. You haven´t written the recipes of your buffers, but I guess that your pre-perfusion buffer is a PBS-like buffer, and your wash buffer has BSA on it. So, I did I first pre-perfusion with PBS, then a second pre-perfusion with wash buffer, and finally the perfusion with collagenase. BSA decreases the activity of collagenase, so its presence does to some extent protect the hepatocytes, while still allowing the digestion to proceed. These are some references of people using this technique, with that wash step added: Gastro, FASEB
As for the rest of your protocol, it looks good; it should work. Only remember to pipet the cells with EXTREME caution and care. Good luck!
Thank you badcell.
I finally had 97.5% cell viability!
Do you have a protocol/reference for coating dishes with fibronectin?