I have tried the attached protocol to isolate primary hepatocyte from mice, using Collagenase instead of Liberase.
Everything is the same as mentioned in the protocol. Except the following :
- Used isoflurane anesthesia 5% for induction and 1.3% for maintenance, no heating pad or lamp.
- perfusion flow rate :7ml/min
- 39mL perfusion buffer and 39mL digestion buffer.
- Cannulated the Inferior vena cava.
The pH and temperature of the buffer is 7.4 and 37C respectively.
Cannulation goes well and liver gets digested properly.
But the cells are all dead immediately after harvesting liver
I want to open it up to a discussion because I am desperately finding out what exactly is going wrong for such low viability. Can someone help me out?
Hello Bhargavi Vk
Some of the reasons that come to my mind are listed below.
1. Low cell viability could be due to either incomplete digestion or over digestion. Poor perfusion can lead to incomplete digestion and therefore low recovery of cells. This will lead to the inability to mechanically dissociate the liver into a single-cell suspension. You should ensure that perfusion using perfusion buffer results in a pale, slightly swollen liver before you introduce the digestion buffer. Excessive perfusion and digestion will lead to over digestion, and it should be avoided to prevent cell death. If you feel that there is an over digestion, you should lower the speed of perfusion and digestion. I feel perfusion flow rate should be reduced from 7ml/min to 2-3ml/min.
2. The digestion will depend on the specific collagenase activity in the digestion buffer. The time of digestion must be optimised due to lot-to-lot variability in collagenase activity. Digest for 10–20 min at 2 ml/min. This will use nearly 20–30 ml digestion buffer in total. When 2–5 ml of the perfusion buffer is left, add the digestion buffer to the 50 ml tube without stopping the pump. Please note that the exact time for optimal liver digestion will vary slightly between mice. The signs of under digestion will include inability to mechanically dissociate the liver into a single-cell suspension. On the other hand, the signs of over digestion will include complete digestion of the liver into a homogenous solution before transfer to the petri dish. Digestion is successful when there are no visible liver pieces after mechanical dissociation, and which can easily be passed through the 70μm strainer.
3. Percoll is used for density separation of the viable hepatocytes from dead hepatocytes and cell debris. There is a possibility that there could be a low cell recovery during percoll gradient cell isolation even after a successful perfusion. This problem could arise when the temperature of percoll solution is incorrect or when too many cells are added to the gradient. Keep the percoll medium at 20°C–24°C to maintain accurate density and to keep the pH between 8.5–9.5. Centrifuge with low acceleration and low brake to minimize trauma to hepatocytes during percoll density separation.
You may also refer to the article attached below for the protocol.
Article Isolation, culture, and functional analysis of hepatocytes f...
Hope this is helpful!
Best.