Does anyone family with zone-specific hepatocyte isolation? I want to isolate hepatocyte from central vein region and portal vein region respectively. I found some papers use digitonin/ collagenase perfusion system, but it is not so stable and complicated. Does any one have good advice or protocol for zone-specific hepatocyte isolation? Thanks a lot!
Sometimes when the protocol used as a reference does not work, one has to modify it to specific needs. The PI supervising the project must have the most appropriate suggestions, if that is not the case, you may be in the wrong lab.
Dear Chao,
it will be very difficult to isolate hepatocytes from the different zones separately. Whenever you digest with enzymes (collagenase, pronase etc.) you will get a mix of both cells and when you plate them you will get a uniform culture. This is because the zonation in the liver is the result of the different gradients of nutrigens, oxygen and pressure. All these factors get lost once you isolate the cells (from any zone). The only thing I could imagine is to dissect the regions first and digest these pieces of liver separately. But this will not be easy and you will probably not get many cells. Or you look for specific surface markers of cells from the different zones and then you sort them by Facs or by magnetic beat columns.
Hi Chao,
I agree with Katja. The solution should be to digest separately the different pieces of your zones of interest to obtain primary hepatocytes from each zone. This is what we used to do preparing primary hepatocytes from livers which were not whole.
Good luck
Hi Katja & Patrick:
Thanks a lot for your advices. Another question is how do I get the pieces of liver from specific zone? Do you have any advice or good protocols? Thanks so much.
Hi Chao,
You could start reading a basic paper from G De Sousa and the following ones from our team, which also describe cryopreservation of hepatocytes, which could also be usefull.
Thawed human hepatocytes in primary culture.
M Dou et al Cryobiology 1992; 29(4):454-69.
I don't know how it works in your country, especially if you need specific liver parts.. (if you can get from donnors or from surgical resections, aso...).
In our team, we have got human liver pieces in the past from hospital, which is of course strongly regulated and quite rare. The reason why we have developped technique of cryopreservation.
Good luck in your quest..
You can download this article from my page on ResearchGate.
All the best,
Ivan Usynin
Selective hepatocyte isolation from acinar zonal distribution, as pointed out by Ivan's excellent publication is difficult at best, and regardless of which density gradient and/or pre-poisoning method (ie digitonin, with portal or hepatic vein infusion) you use. Once isolated, the zonal differences of in vivo hepatocyte functions will gradually disappear over time in culture (minutes to days, depending on how functional your culture system is, and perhaps what components are present that might favor maintenance of one zonal pattern over another). Density gradient separation will depend on your specific donor model and how they are fed, age, maybe gender, other treatments.
You will want to control all of these parameters, use a couple of assays as suggested by Ivan to 'prove' each enriched isolation is actually enriched, and then perform or use the data from your zone specific experiments based on a predetermined specification of successful zone-specific enrichment. If you can also do organ donor histochemistry to provide added proof that there is a zonal gradient of the markers you use to determine successful zonal isolation, in the animal model you are using for isolations, this will strengthen your argument for any conclusions you make from the zonal isolation studies.
You are absolutely right that in vitro zonal hepatocyte research is a pain, essentially because of the issues you point out. Few people take it on and then a lot quit because of the issues of isolation.
Good luck and strong presistence! Invent a better way and you will be remembered forever ;-)
Hi Block:
Thanks so much for you advice, it is very useful, I will keep it in my mind. And I will try my best to find a better way.
With best,
CHAO
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