Primary rat hepatocyte isolation

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Arefeh
Arefeh's picture
Primary rat hepatocyte isolation

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!

R Bishop
R Bishop's picture
Its really not that hard. I

Its really not that hard. I will upload the protocol later today. Been doing it for years.

Rb

R Bishop
R Bishop's picture
As promised, here's my

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

DBI
DBI's picture
Hi,

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

R Bishop
R Bishop's picture
I used to do it through the

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

DBI
DBI's picture
thanks a lot for your quick

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?

DBI
DBI's picture
So i tried doing it through

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..?

R Bishop
R Bishop's picture
The answer is an emphatic yes

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

DBI
DBI's picture
Thanks! I will do so!

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 )

DBI
DBI's picture
Another issue which i wanted

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?

samm
samm's picture
Have you thought about using

Have you thought about using a collagen+alginate matrix?

DBI
DBI's picture
Hey,

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
 

samm
samm's picture
The alginate basically serves

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
Michal Kfir's picture
Dear Dr Bishop,

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

Xiaoping zhao
Xiaoping zhao's picture
I am working rat hepatocyte

I am working rat hepatocyte isolation to do compounds stability. I just want to know the viability of primary hepatocyte. Thanks a lot.

cell group ente...
cell group enterprises's picture
<br /> is it worth it to just

<br /> is it worth it to just buy hepatocytes from the many vendors out there?

pathos
pathos's picture
The protocol posted by Dr

The protocol posted by Dr Bishop is pretty much standard.  The use of KRB is old school and used by many labs today.  All that is important is to maintain a normal extracellualr saline.  The simpliest buffer is S&M

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        S & M (1X)
Combine the following chemicals, in a beaker; bring volume to 8 L or by adding deionized distilled H2O using a stir bar to mix.  Adjust to pH 7.4.  Filter sterilize using 500 ml Nalgene filtration system with 2 μM pores into 16 glass bottles and store at 4o C.
For 500 mL stock                                    For 8L stock
HEPES                        1.2 g                                                            19.2 g
NaCl                        4.15 g                                                            66.4 g
KCl                        250 mg                                                            4 g
NaOH                        95 mg                                                            1.52 g
 
       100X CaCl2·2H2O
Add chemicals to beaker and bring volume to desired amount using deionized distilled H2O.  Store at 4o C.
For 100 mL stock                                    For 200 mL stock
CaCl2·2H2O             2.94 g                                                            5.88 g
Our lab would make the SM ahead of time and store in a 500 ml bottle which on day of isolation is split into 250 mlx 2 bottles.  Buffer 1  will have EGTA  1-2 mM  or none at all.  the purpose of this is to get rid of Calcium.

These can be used for Mouse Rat or human hepatocytes.  Except with human, glucose may also be needed ( see Strom et al for method)

next is collagenase.  This is the most influential material used in the isolation.  I find Collagenase, Type IV from SIGMA is the best and most reliable.  Also  Very cheap.  Use 25-30 mg /250 ml.  This enzyme is very temp. sensitive so make sure it is 37 C upon entry to animal/liver.  flow rate should be  25 ml/min for rats.  Use all 250 ml of each buffer A and B.  Also if you have pooor surgical skill then avoid portal  use the inferior vena cava.  For this u secure catthater to inferior vena cava.  when flow starts cut  up the through ribs and clamp the superior vna cava and then cut the portal.  this will perfuse liver in a retrograde direction relative to normal  blood flow.  after all solutions have been used then carefully exceise liver and filter over nylon mesh (100-200 um pore size) and use COLD  SM (4 C) with 1% BSA to wash the cells.  Spin 3 timesat 50-60g for 5 min each.  Cells should be 80-95% viable.

For the other cells of the liver, look in the supernatant from these spins, they will be there.  Generally, centrifuagal gradients will be needed.  I used to use an centrifugal elutriator. 

Viability is determined by trypan blue methods. 

Good luck

pathos
pathos's picture
One can not reliably buy

One can not reliably buy fresh hepatocytes.  Most hepatocytes sold are cryopreserved and have serious issues with attachemnt to plates.  phase 1 and 2 conjugation systems still work so these are used for tox studies by drug companies.

leo28
leo28's picture
I have been trying to culture

I have been trying to culture mouse hepatocytes. I am able to isolate the cells also. The problem lies in their adherance. the hepatocytes are not adhering to either collagen or matrigel and come off very easily upon very mild wash with PBS. please suggest.

thanks

drgaju.15
drgaju.15's picture
is this forum is still active

is this forum is still active? i have some doubts regarding rat hepatocytes and RBC.
how they look like under light microscope in trypan blue?

thans

pathos
pathos's picture
Rat hepatocytes are about 18

Rat hepatocytes are about 18 um   and RBC is about 4 um.  If you can not figure out which is which then lok for the nucleus.  Hepatocytes have one big one, likely more.  27 % of rat heptocytes are binuclear.

If mouse cells are not attaching then Uyou likely used to much collagenase.  The collagenase btw is not one enzyme.  It has thermolysin as well.  Sometime refered to as neutral protease.  It is required to release hepatocytes but DEADLY to them if exposed to long.  Step 2 of Seglen method is saline with collagenase.  make sure you digest no more than 10-12 mins.  Look at the Glisson capsule if you see it looking mushy STOP and filter cells  

Good luck all

BIOLOGISTE
BIOLOGISTE's picture
hi, when we isolate

hi, when we isolate splenocytes from the spleen we dont use collagenase, can i delete the collagenase etape and isolate hepatocytes without using collagenase

drgaju.15
drgaju.15's picture
hi

hi
i perfused rat liver successfuly got hepatocytes but vaibility is aroound 25 to 30% only. can any body help me how to increase viability. useing heps buffer DMEM with collagenase for perfusion, PH 7.2 @ 7ml/min.

thanks

sonidurgesh
sonidurgesh's picture
Which protocol you use

Which protocol you use

Sel
Sel's picture
Dear Pathos,

Dear Pathos,

I've tried to perfuse and isolate Rat's hepatocytes (we use Spraque Dawley rat) following your protocol. So far I could manage and perform complete perfusion process in 15-20 mins. But now I'm confused how the viable cells exactly look under microscope in trypan blue? I got mostly quite big cells with bright and shiny cytoplasma but with blue nuclei. Do you consider it as a live or dead cells? Do you have any pictures of rat hepatocyte that you can show me so I can compare it with mine. Or I can send you my cells picture if you allow me to pm you. What do you think about chemicals that we can use to determine the viability of the cells like MTT assay or Cell Counting Kit like CCK-8 from Fluka?

Thanks and regards

Adel
Adel's picture
Please contact me and i will

Please contact me and i will help you (by e-mail)