The standard method for isolation of primary normal rat hepatocytes is to cannulate and perfuse the hepatic portal vein first with Hank’s buffer for washing followed by collagenase in digestion medium to breakdown the elastic structure of the liver. The liver is then removed and again soaked with digestion medium and collagenase.
Can I first remove the liver aseptically followed by soaking in Hank’s buffer, then in collagenase for a longer period of time to dissociate cells by mechanical shredding, and finally filtrate through a 70 µm mesh?
There is no good alternative to the 2 step perfusion method ... except for the fetal liver where you can use the method you suggest
Many years ago I published a method adapted from earlier work, in which the liver was removed, finely sliced and the slices incubated in collagenase solution prior to washing and filtration. The method can be found at Analytical Biochemistry (1976) 71, 341-350. The yield is less than with lobe perfusion, and the technique takes longer. It works better with liver from younger animals.
This may help you.
I am afraid that the proposed alternative won't be very effective as you can't optimally reach all hepatocytes with collagenase and you might harm them too much by mechanical shredding.
To make the perfusion easier, you can also use the vena cava below the liver to cannulate, it is much bigger and easier to hit. At least this is what I experience with mice, I haven't perfused any rats yet.
i used the attached method, and got after a little practice, a cell viabiltiy of around 80-90%, but i don't know if this will be less work, but this method didn't need a perfusion.
The alternative method which suggested by the author not effective as standered method where their is probability that many hepatocytes may be lost in the alternative method. In spit of this, a laboratory trials for comparison between both methods must be undertaken by the author to measure the yield of hepatocytes in both.
-The young aged rats have active and relatively free cells from fate and fibers which l help in rapid and effective yield more hepatocyte .
NO PROBLEM TO THE AUTHOR TO UNDERTAKE BOOTH METHODS TO REACH THE GOOD ANSWER OF HIS Q AND ALL UNDERTAKEN METHODS DEPENDED ON OTHER FACTORS IN LABORATORY CONDITION AS HUMAN SKILL, ACCURATE APPLICATION , AND USED REAGENTS
MUCH OBLIGATED
Alternatively, you can remove the lobes and perfuse the lobes. This is a standard procedure for human liver.
FOR ALL COLLAGES
The scientific research not limited to any methods but continuous trials in laboratory will help for answers to all my questions. In general, the researcher must give the chance for application all available methods and can suggested a modifications of all old methods .Otherwise, the author can use 2 methods for comparison and investigated a new alternative method with modification of the standard method.
CONTINUOUS INVESTIGATION OF MODIFICATION IN THE OLD METHODS MUST BE UNDERTAKEN FOR RECENT APPLICATION.
THE SCIENCE NOT KNOW THE LIMITATION IN METHODS WITH THE CONTENTIOUS, SUCCESSFUL TRIALS .
Much obligated
In my experience, yes, you can, however the efficiency is much worse than by using the standard method. So, depending on your aims, maybe you can skip the perfusion step. In my protocol there is a last step which I use Percoll. It Works fine with me. Good luck.
I second the use of percoll for a final clean-up of the prep by removing the vast majority of dead cells.
Your viable cell recovery will be lower per weight of liver when removing the perfusion step. Perfusion of the liver was included to removed endogenous proteases that are released that cause autolysis (dead and dying cells). If healthy viable cell numbers are not a concern, I would agree with the others.
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