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Primary rat hepatocyte isolation

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Arefeh
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Topic Started by Arefeh
on 12/4/2008 2:38 AM   
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I want to start a new project, a kind of 3D culturing of primary rat hepatocytes...so I have to isolate them from Rat Liver, Does anybody have experience about it?The protocols mentioned in articles seem very hard!


Last edited Feb 15, 2009, 5:42 AM by Arefeh
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R Bishop
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Posted By R Bishop
on 1/6/2009 9:21 AM   
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Its really not that hard. I will upload the protocol later today. Been doing it for years.

Rb

"Everything should be made as simple as possible, but no simpler."
-- Einstein



R Bishop
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Posted By R Bishop
on 1/6/2009 10:30 AM   
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As promised, here's my protocol. It took me about 6 months to perfect this protocol. Should work exactly the same for the rat.

Primary Hepatocyte Isolation Protocol

Please let me know if you have any specific questions. For some links to publications that I used this protocol in check these

Cell and Microbe Paper 2008

Rb

"Everything should be made as simple as possible, but no simpler."
-- Einstein



DBI
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Posted By DBI
on 6/29/2009 11:09 AM   
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Hi,

thanks for the info. I am just getting started with hepatocyte isolation with no luck so far. I think you are perfusing through the heart. Most people do it thorugh the portal vein I guess but I found to do it easier through the heart as (eventhough it is a rat that i am working with) the vein is really difficult to see. Is perfusion thrugh the heart trickier in terms of cell viability? or are there any drawbacks?

AA

AA



R Bishop
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Posted By R Bishop
on 6/29/2009 11:47 AM   
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I used to do it through the portal vein, but I ruined about 1 out 3 animals with my lack of technical skills of inserting the catheter.  Thats why I went to the left ventricle with thwe catheter and clipping the portal vien method.  I actually got better cell viability in mice.  Its been a LONG TIME since I worked with rats, but I see no reason the same wont work.  Does the liver clear rapidly when you perfuse through the heart?

 

Rus

"Everything should be made as simple as possible, but no simpler." -- Einstein



DBI
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Posted By DBI
on 6/29/2009 12:05 PM   
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thanks a lot for your quick response! So last time (my second trial) we started of with portal vein but somewhere along the procedure we lost the vein so we decided to go through the heart. It did get slightly pale but i am not sure if the person which was helping me out did it form the correct lobe. I think we will try the heart next time as you described it. I will see how that goes. we actually do deep anesthesia with isoflurane that may also be affecting the perfusion. By the way, i am also looking for a bubble trap which can take 30ml/min flow rates. Do you guys make your own trap? if yes can you explain how? i saw only Seglen explaining the silicone tube with cotton inside but is there anything more advanced out there?

AA



DBI
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Posted By DBI
on 7/14/2009 16:27 PM   
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So i tried doing it through the vena cava and cutting portal vein method.( I didn't try the heart yet ) The kidney burst during the procedure although we had clamped the needle. The color of the liver did eventually change a bit but it was not fluffy or anything like that. At this point i am not even sure if it is the collagenase that is causing the problem(sigma from clostridium..C5138). I think i am still having trouble with the path of the solution.  Do you think it makes sense to practice with a saline solution first to get the basics of the needle insertion? Because if the path is correct the liver should get pale even with the saline solution only.. right..?

AA



R Bishop
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Posted By R Bishop
on 7/15/2009 8:23 AM   
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The answer is an emphatic yes!  You definitely should practice with any animals that the lab can spare or simply order up some inexpensive rats and get to it.  You are performing a fairly complicated surgical procedure on a pretty small animal.  I went through a lot of practice mice before I could even get one liver to clear properly.  You want that liver to clear immediately to a pale tan color with no streaking or red splotches.  If the path is wrong then that will not happen.

 

One thing I might suggest is to take a look around your university or company and find people that are experts at small animal surgery.  They can teach you a lot.  The hard part is inserting the catheter without puncturing the vena cava or portal vein and then clipping the opposite one without bumping things etc. Thats why I started perfusing into the left ventricle and clipping the portal vein.

 

RE: your 6/29 post I never used a bubble trap and I also sac'd the mice with isofluorane just prior to perfusing.  The hepatocyte viability is really all about speed of getting the perfusion done, proper collagenase concentration, and good sterile technique.  30ml/min?  That is a really fast flow rate.  I might back that off a bit.  I had a great success with 7ml/min.

 

The Bishop

"Everything should be made as simple as possible, but no simpler." -- Einstein



DBI
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Posted By DBI
on 7/16/2009 9:12 AM   
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Thanks! I will do so!

I am working with ~150 g rats. That is why i am using ~30ml flow rate. I know it is kind of high and Hepatocytes don't really like shear force a lot i guess. I can decrease the rate a bit next time.

For the bubble trap, i started using an IV infusion set with a drip chamber (Kawasumi   Iv Administration Set - Vented 20dr/Ml Y Site 84 )

AA


Last edited Jul 16, 2009, 11:14 AM by DBI

DBI
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Posted By DBI
on 7/20/2009 6:37 AM   
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Another issue which i wanted to discuss is 3D encapsulation of hepatocytes. SO my ultimate goal is the encapsualte these cells in a synthetic peptide hydrogel 3D. I assume that they must be pretty fragile after the whole isolation process. I am not sure how to proceed after the isolation process. 2D should be no problem as it is staright forward but with 3D it gets complicated. I can not use any BSA or FBS or that kind of big proteins since those will interact with my peptide and prevent the assebly of the hydrogel to occur. Any suggestions?

AA



samm
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Posted By samm
on 7/20/2009 7:49 AM   
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Have you thought about using a collagen+alginate matrix?



DBI
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Posted By DBI
on 7/20/2009 8:00 AM   
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Hey,

i can not add those since it will interact with my peptide system...I need to suspend them in cell culture media but somehow condition them before encapsulation so that they survive the process of encapsulation. In terms of matrix i have my own peptide which i synthesize, using collagen or alginate would only be useful as controls... I am looking for a cytoprotectant which works well with normal hepatocytes

 

AA


Last edited Jul 20, 2009, 11:12 AM by DBI

samm
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Posted By samm
on 7/20/2009 11:57 AM   
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The alginate basically serves as a neutral, better-defined scaffold for 3D culture - if you have your own matrix, thats even better than using an undefined collagen system! Also, the alginate system relies more on carbohydrates than proteins - still has a weak charge, but might work out betterfor the peptide you are testing.



Michal Kfir
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Posted By Michal Kfir
on 7/14/2010 3:55 AM   
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Dear Dr Bishop,

I would like to ask you several questions regarding your primary hepatocyte isolation protocol.
My initial aim is to isolate kupffer cells and hepatocytes from mice and  take these cells (as is - without tissue culturing) to RNA and protein analysis. My questions are:
1. Do you have any experience with separating livers to the different cell types (mainly kupffer vs hepatocytes)? 
2. Have you tried isolating hepatocytes without perfusion?  I thought that maybe in my case (no culturing) i don't need very high efficiency.
3. We don't have here a centrifuge for 50g (the minimum is 300rpm) any idea how to overcome this issue? 

Many thanks,
Michal




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Posted By Xiaoping zhao
on 7/22/2010 23:32 PM   
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I am working rat hepatocyte isolation to do compounds stability. I just want to know the viability of primary hepatocyte. Thanks a lot.



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