Hi, 

I isolate mice primary hepatocytes too. My viability is much higher than your reported 50-70%. Mine is >90% viable. I also usually pool hepatocytes isolated from at least 4 mice and can get up to 

Hi, 

I isolate mice primary hepatocytes too. My viability is much higher than your reported 50-70%. Mine is >90% viable. I also usually pool hepatocytes isolated from at least 4 mice and can get up to 

Hi,

did you receive a new collagenase? Sometimes when we ordered a new one we also got a new LOT number and then the perfusion time had to be adjusted again.

Best,

Anastasia 

Yes, I will go with Anastasia..Collagenase lots are usualy the problem..U can try adding trypsin inhibitors , if that helps..or decrease the digestion time/collagenase amount..that may solve the problem..also another factor can be the flow rate and pressure..check those and play with those as well

Hi,

For the low cell number, collagenase is most likely the source of the problem. For the cell viability  the pH of the perfusing solution is very important. You have to take into account that the pH changes during perfusion and that the perfusing solution needs to be prepare to account for this change.  Good luck.

Hi Lamming

You can check each step in the isolation procedure to know about the hepatocyte quality. Every step indicates whether the next step will be successful or not.

Since you are getting good viability, we can assume that the perfusion, collagenase concentration, enzyme activity, perfusion speed, temperature, duration and cell retrieval from liver are all okay. 

I doubt the position of liver while perfusion.  All lobes are not correctly perfused.  Hold the liver by portal vein to just lift and place it back slowly so that the lobes spread on surface without twisting and crossing.

Wish you all the best for the next isolation

I've had problems with low cell number  because cells are lost at the percoll spin. We would overlay cells on the percoll  and spin them through but if the cells aren't initially put just below the top surface of the percoll they might have a hard time getting through.  Changing this step easily increased our cell numbers by 10-20%.

good luck! 

Hi,

I have experience in isolating rat hepatocytes and usually low yield and low viability (50-70% is low) together is due to poor instead of excessive digestion.

Is the liver properly digested? Is it soft and release all the content easily when disrupting the Glisson´s capsule? or it is hard and is very difficult to disrupt?

Poor digestion can be due to many causes as already stated by others such as a new lot of collagenase (reset quantity and time), improper temperature, oxygenation, presence of Calcium in the digestion buffer, etc.

Hi,

I think the viability can be improved a lot. In rat without using percoll I used to get viability > 85 % consistently. One point sometimes people ignore is dissolving the pellets. Try first in very small volume of medium, then gradually go to higher volume while dissolving.

I agree with the previous answers that the quality of the Collagenase is critical but that problem usually shows up as REALLY low viability.  It sounds trivial, but I never did get accurate cell counts on primary hepatocytes because they clump so badly.  Most of my cells wouldn't show up under the hemacytometer slide (if that is how you are counting them).  I suspect my viable cell counts were always an under-estimation.

To remove clumps one of the important most step is the cells must be in suspension ..thus dissolving the pellet first in very small vol of medium helps a lot

Hi Malden,

In my hands I always obtain single cell suspensions which you can see as perfectly separated vital cells on a hemocytometer under microscope. If the collagenase digestion is poor you will get clumps of cells and if it was too harsh you will get dead cells.

I agree with Sagnik that the cell pellet should be first completely resuspended in a tiny amount of liquid (buffer or medium) and then brought to the desired volume. If you have a pellet without any liquid on top and you add the final volume of buffer to it, the pellet is very difficult to resuspend (seems almost like a rock).

Regarding “REALLY low viability” I must say that, when attempting to damage hepatocytes with chemicals I could never get viabilities under 30-40% (as measured by Trypan blue exclusion or LDH release) so, in my experience, hepatocytes are not that easily killed.

Small numbers but good viability usually means incomplete digestion. But you have to balance this knowing that too much collagenase time is not good either. Gentle resuspension (as mentioned above) will help get more single cells. Remember you need single cells for plating in culture. Clumps are not good.

For rat hepatocyte perfusions, our lab easily gets >95% viable. Of course, rat also gives you a ton of cells! Mouse perfusion is tricky. Even with the most experienced person in our lab (20+ years), mouse hepatocyte viability is still 60-65%.  I also think that mouse hepatocytes seem more fragile and susceptible to breakage with too much manipulation.

Good luck!

I am a retired person, and, when teaching students, have experienced several troubles to get high yield of hepatocytes from perfused mouse liver. I would like to make some comments to your question.

Simple but the most serious trouble;

My student, who could get poor number of hepatocytes, did not thoroughly mix the perfusion solution(Hanks(Mg-free)). She wanted to prepare one liter of the solution in one liter-mess cylinder(long-type). The reagents were easily dissolved, so that she sterilized it with a filter, which was set on the 500 ml bottle. She got two bottles with sterilized Hanks. Collagenase(We used Sigma collagenase.) was dissolved in the Hanks solution. But she got poor results. I tried several times using Hanks solution, which she prepared, and got the same results. Finally we found that the Hanks solution was not good. Although the reagents were surely dissolved, concentration of them in the upper phase of the cylinder was different from that in the lower phase. We could constantly get high number of the hepatocytes using thoroughly homogenized Hanks solution.

I would like to add more comments.

I feel that mouse hepatocytes are more fragile than rat, so that rapid preparation is recommended.

It is not good to observe clumps of hepatocytes after Percoll centrifugation. Check the concentration of the collagenase.

After Percoll centrifugation, the viability is usually more than 90 %.

If you want to cultivate mouse hepatocytes in serum-free medium, several commercially available one are not inappropriate. I tried five medium and Waymouth was the best one for my studies on expression of drug-metabolizing enzymes. If you want to cultivate them in serum-containing medium, you do not care it.

If so, you need to concern increasing dosage of liberase or collagenase type IV.

At the end of the perfusion process, transfer dissected liver tissue to beaker (pre-warmed HBSS) and gently shake ten time. Then, add stop buffer.

Good luck!!

It is also important that you don't push cannula to much inside (i.e. Intrahepatic portal vein) which will cause incomplete perfusion. Try to fix cannula at extrahepatic portal vein.

Sorry but 50-70% of cell viability is really not good, especially for such a small number of cells.

What enzyme(s) do you use at which concentration(s)?

How long is the liver digested and how do you double-check that the liver is digested?

Do you spin your cell suspension?

I want to have an idea or what you are doing to see where those issues are coming from

hi 

i'm facing difficulty in isolating hepatocytes from rat. before adding percol i got around >90% viable cells but after adding percol ALL i got dead cells?? the perfusion looks good also i can see the signs of digestion.

i tried that many times and do not know whats wrong.. i'm using the same protocol my colleague used and he but not me got the cells. 

any suggestions please :(...

If you have 90% viable cells, why would you use an ulterior pecol gradient? With sieving through a double layer of gauze and low speed (50 g) centrifugation you will get hepatocytes free of cell debris.

If you do not submit your preparation to percol, is the viability maintained for a few hours? or it is lost within a short period of time? I am asking this because the lost of viability can be due to an excess of collagenase concentration or very prolonged digestion.

Could anyone please provide the recepi and method for percol centrifugation ?

How much percol (mine is sigma Sigma P4937-100ML) and how much cell solution do I need ?

Thanks a lot.

Riccardo

Could anyone suggest me a good collagenase? My old bach is no longer available and I don't know  which one is better!

Andrea I would suggest collagenase Type IV from Sigma (cat # C5138) for hepatocyte isolation, this worked well for us

Hi Dudley William,

If the viability is good, then I would suggest a check on perfusion.

1.  Temperature of perfusion buffer - 37C.

2.  Flow rate of perfusion - 20 ml/min

3.  Proper oxygenation - Oxygen bubbling.

4.  Spread the liver lobes to ensure proper buffer flow inside.

5.  The duration of perfusion with Ca free buffer before collagenase perfusion shall be increased.

6.  Collagenase shall be freshly prepared.

Regards

Anil

thanks Joshua!

Hi Everyone!

Thanks for your answers.

With younger mouse (7-9 weeks old) I was getting good perfusion and the liver was turning pale but with older mice (9 months old) my protocol is not working :(

I tried increasing the perfusion and digestion time but could not achieve success :(

Also, does anyone have any idea about number of hepatocytes that could be isolated from 9 months old mouse ( C57BL/6 mouse)?

Please let me know if you also have any literature or protocol to support your answer.

Best regards,

Ankur

can anybody please share the complete protocol for primary mice hepatocyte isolation, because I am also facing the same issue of poor viability, I am using collagenase D of 1mg/ml concentration in digestion buffer, which is HBSS..Flow-rate of perfusion and digestion is 3ml/min. In digestion buffer HBSS, we have Calcium, but perfusion buffer we dont use Ca.