There are tons of protocols for hepatocyte isolation from whole livers and lobectomies, and they all depend on perfusion with a collagenase solution. My group is collecting wedge biopsies from the margin of the left lateral section. Our goal is to isolate hepatocytes from these biopsies for hepatocyte transplants into a murine model. we never get a suitable vessel for perfusion.
Here's what I've tried so far:
1. slice tissue thinly with a #10 scalpel.
2. Incubate tissue in collagenase/dispase at 37'C for 30 minutes.
3. gently mash tissue through 70um nylon screen with syringe plunger.
4. Spin cells on 35% percoll gradient at 30 x g 5 minutes.
5 check count/viability by trypan blue exclusion on hemocytometer.
I get plenty of hepatocytes, but they don't look good. Many are either blebbing or have clearly compromised membranes. I think the warm ischemic time during the long digestion is to blame for the former, while the nylon mesh may be responsible for the latter.
Is anyone aware of a better way to do this?
You can improve pass 1. You need smaller pieces of tissue. Use a gauze and comprime manually to obtain a cell mass before the incubation with collagenase.
Another option ir to avoid percoll gradient. Blood cells grow in suspension and in a few days you can eliminate them just changing media. If media turns yellow after 24 hs, dont change media, just add more fresh media.
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