mouse hepatocytes isolation

[amicia]

Dear members,
Could anybody help-me? I'm looking for a mouse hepatocytes experts becasue I have some problem to isolate very good mouse hepatocytes. I'm doing a two-step isolation but I'm not able to recovery viable hepatocytes.
All suggestions are welcomed.
Many Thank

[moleculardude]

Hello Amicia!!
how are you?

We are experts for mouse hepatocytes isolation. I can post a protocol soon.

Briefly we use infusion and permeabilize the liver first. This breaks up any connective tissue in the liver. We then separate out the liver into primary hepatocytes and plate them.

Look for the protocol soon.

[amicia]

Dear Moleculardued,
I'm very happy to konw the experts in the field of mouse hepatocytes isolation because so far I spend four mounths of my life in order to obtain good hepatocytes but I failed.
Briefly I'm isolating hepatocytes with a two step perfusion method, but I'm perfusing trough the heart and not trough either portal vein of inferior vena cava. At first I perfuse with a EGTA (0.5 mM) solution for 2-3 min and after I perfuse with a collagenase solution (0.05%) for 5 min; thein I excise the liver and I minced it into with scissors on ice in a petri dish in a buffer with the same composition of collagenase buffer but w/o collagenase. After that I recover the brief and the solution with a large bore pipet and I filter in a nylon mesh (50 micron), finally I centrifuge twice for 2 min at 50 g with the same buffer. Then I resuspend the pellet in a william E medium supplemented with 10% FBS and insulin. My problem is when I look the cells with trypan blue stainning the 100% of nuclei of my cells are blue, so the cells are die!!!!! Of course when I plate them they do not attach very well and after two days they don't form the typical structure of hepatocytes in vitro. I'm not able to perfuse trough the portal vein or inferior vena cava because is really difficult for me to cannulate the veins. Anyway the liver is very well perfused and partially digest when I put it into the dish. So do you think perfusion trough the heart can induce a release of some substances able to stress my cells?
Could give you my protocol so you can give me more detailled suggestions?
Thank you in advances for your kindness and hope to hear from you some news as soon possible.

[kiki06]

Hello Amicia,

your protocol looks good although from what I know of experiments going on in a nearby lab, they perfuse directly into the liver.

If you get 100% dying cells that is defininetely not good. mouse is harder than larger animals so you must you very fine needle / catheter to do this... although it is done by the lab i mentioned...

just some infos... hope it helps

[moleculardude]

Yes please post your protocol these are some tips from a lab we are close with:


- butterfly needle 25G for mouse perfusion

- insert butterfly needle into abdominal vena cava
- sever portal vein to allow outflow of perfusion
- add perfusion media
- add collagenase digest media
- both are pumped into the liver slowly
- cut the diaphragm to sacrifice the animal
- remove digested liver
- harvest into cell suspensions
- assess viability with trypan blue assay
- wash cells (centrifuge very slowly)
- plate onto flasks
- if they stick and look healthy you did well
- currently labs get 80-90% viability
- cells can last about a day or so

Many cells begin apoptosis as soon as they lose cell adhesion contacts:
http://www.ncbi.nlm.nih.gov/entrez/q...&dopt=Abstract

this means you will need to be quick to plate them asap!


a protocol for mouse:
http://www.pubmedcentral.gov/article...gi?artid=86995


other protocols
http://www.cellzdirect.com/CD_Public...st_10-2005.pdf

another good protocol for rats:
http://www.wjgnet.com/1007-9327/10/699.pdf

[amicia]

Hello Moleculardude, Hello kiki06
happy to hear news from you about my problem. At first many thanks for your suggestions, me too I think that the problem is during the perfusion, may be is not possible to perfuse trough the heart because during this operation some drugs, hormones or other are been release into the circulation and could induce apoptosis on my cells. I knew two of the papers taht Moleculardude suggested me, and in both cases they did vein perfusion. So I'm moving in this direction. Hope to be able.......
Anyway thank you a lot for your help, and if you have more suggestions, don't hesitate....
With best regards

This is my protocol:
1. Worm the collagenase type I 0.05% (Sigma, add the collagenase immediately before the use to a Buffer with HEPES, NaCl, KCl and KH2PO4, as reported in the paper of Seglen: Seglen PO. Preparation of isolated rat liver cells.Methods Cell Biol. 1976;13:29-83.) and another buffer with the same composition but w/o EGTA and with calcium.
2. Flow trough the pump until the pipes are completely filled of buffer A. Take 10 ml of Buffer for the collagenase digestion but w/o collagenase (separation buffer) and put into one Petri dish.
3. kill and fix the animal on the appropriate table:
• After washing with ethanol (70%, to avoid mycoplasma and hairs contaminations) the ventral of the mouse, make a small cut at the level of the pubis, such as to allow the entrance of one point of the scissors.
• Perform a median longitudinal cut superiorly to the chin, having cared to carefully remove the skin and the underlying musculature.
• Open with scissors the left thoracic cavity starting from the last rib by cutting the costal cartilages at their point of union with the bone. Continue cutting, until uncovering the pumping heart.
• Put the catheter (25 GA 0.50 In) of the pump into the heart (left ventricle), start to perfuse with washer Buffer (with EGTA) and at the same time cut the right atrium; when the liver became white (2-3 min) stop the pump and put the tube of the pump into a falcon 50 ml containing the collagenase type I in order to avoid any bubble formation into the tube. Switch on the pump continuing to perfuse for 5 min or more until the liver softening of the tissue gradually appears as well as marbling, finally the liver tissue appears like as sponge (as marker prick the liver and note the formation of white spot and the consistence of the tissue), consequently you have to regulate the speed of the pump in order to digest the liver very well. When perfusing put the catheter into the left ventricle (the left ventricle is under the right ventricle when the mouse is prone on the back; so is need to raise just a little the heart to start the perfusion) and cut the right atrium in order to flow the blood from the animal. Remove all the liver and put it quickly into the Petri dish containing separation buffer. Then cut it in small pieces with a scissors at room temperature. During this phase the pieces of liver release small particles (cluster oh hepatocytes)
4. Place the dish on ice to arrest the collagenase digestion.
5. By using a plastic pipette (Falcon, 3 ml) aspirate the media and filter it trough the mesh filter (50 micron), add 5-6 ml of separation buffer to the dish and aspirate and inject the pieces of liver very gently. Avoid aspirating and injecting biggest pieces of liver in order to not stress the cells very much. Repeat the procedure 4-5 times. If more than one mouse is sacrificed, put the tube containing the cell into the incubator.
6. Centrifuge the cells at 50 g for 2 min at room temperature.
7. Aspirate the separation Buffer and wash the pellet with the same buffer and repeat the centrifugation (this step is to be sure about the elimination of collagenase from the cells).
8. Discard the supernatant and dilute with fresh medium the cells in an appropriate volume.
9. After leave the cells to attach to the bottom for 4 hours or 1 day and then replace the medium. Sometimes when replace the medium we can detach the cells, so another procedure is to add fresh medium without removing the old medium, bur in this case be warning to the amount of medium into the well.

[kiki06]

Originally Posted by amicia

....and at the same time cut the right atrium; when the liver became white (2-3 min)...

Hi Amicia,
this maybe your problem as you are losing blood supply to the liver and cells are quickly beginning to die from what I know. I would try at least a few times with fine 25 to go through mouse vein directly to the the liver. This way at least the liver still gets blood and is fresh when taken out.

This minimizes apoptosis and death of the cells. after all even if you get a few to stick, they may not be healthy.

Try the portal vein / vein to liver method... maybe you will get much better results... i can ask about the heart perfusion...

talk to you soon
Kiki

[amicia]

Hi Kiki06,
after the launch i'll try trough the inferior cava vein, because the portal vein is really too narrow. I tell you after....., hope.......
Many thanks Kiki

[amicia]

Hello guys,
with the help of a magic technician i perfused trough the portal vein, 3 min with egta buffer and 7 min with the collagenase buffer, but.........., finally the cells present the same shape and a lot of them are stainned with the trypan blue, so I don't what I have to do now. All suggestions are welcomed

[admin]

Hi Amicia,

Hmm tough stuff.... I will get some ideas from a close friend which does amazing isolations on primary hepatocytes... hope that we can help you soon.