Currently I am investigating the effects of knocking down Pex-5 from hepatocytes using primary hepatocytes from WT and liver specific Pex-5 KO mice livers. As these primary hepatocytes are difficult to culture for longer time periods as well as to transfect with some plasmid, I am thinking of using a hepatic cell line in which I can knockout Pex-5 using siRNA and then do the same experiments in these cells. Please suggest me the best available option.
I had used HepG2 and HepaRG and both work well in transfection especially HepG2.
Hepa RG (http://www.ncbi.nlm.nih.gov/pubmed/12432097) is more relevant because they are near to PHH but they are long and difficult to abtain. SO perhaps you should use HepG2 cells!
hope it will be useful
Boris
Thank you very much Dirk and Boris. I will look it up further and then decide.
Regards,
Abhijit
We always use HepG2 cells for transfection assay, high confluent cells worked better.
Perosnnally i used differentiated HepaRG with DMSO and my siRNA from Qiagen worked very well. However HepaRG and HepG2 cells are cancerous (Hepatocarcinoma derived cells).
Moreover, if you need exclusively Non-Cancerous cells, you can buy Human hepatocyte form ScienCell Research Laboratories (Human Hepatocytes (HH)
Catalog #5200 ). Hope that it will work ! Good luck
Hereby some references :
[1] Runge, D., Michalopoulos, G. K., Strom, S. C. and Runge, D. M. (2000) Recent advances in human hepatocyte culture systems. Biochem. Biophysi. Res. Comm. 274:1-3.
[2] Chen, H. L., Wu, H. L., Fon, C. C., Chen, P. J., Lai, M. Y. and Chen, D. S. (1998) Long-term culture of hepatocytes from human adults. J. Biomed. Sci. 5:435-440.
[3] Fitzpatrick, E., Mitry, R. R. and Dhawan, A. J. (2009) Human hepatocyte transplantation: state of the art. Intern. Med. 266:339-57.
[4] Okamoto, M., Ishida, Y., Keogh, A. and Strain, A. (1998) Evaluation of the function of primary human hepatocytes co-cultured with the human hepatic stellate cell (HSC) line LI90. Int. J. Artif. Organs 21:353-359.
You might conisder the HHL cell lines described in this paper:
Liver cell lines for the study of hepatocyte functions and immunological response.
Clayton RF, Rinaldi A, Kandyba EE, Edward M, Willberg C, Klenerman P, Patel AH.
Liver Int. 2005 Apr;25(2):389-402.
PMID: 15780065
I used them briefly when I was a graduate student. I think they would be useful for your current study.
I tend to agree with Eva, primary cells make the most sense to me as a choice, even though they are harder to work with.
You say you're looking at the effects, how? Are you measuring gene expression or cellular responses like ATP, caspase, ROS, etc.? Particularly if you are using cellular response as a correlate of hepatocyte functionI think the metabolically relevant cell population is best where feasible and so would suggest primary hepatocytes.
Thank you Eva and Reagan.
Yes Reagan I am interested in looking at cellular responses like ROS and ATP. Apart from that I am also interested in looking at the changes in mitochondria by EM analysis and fluorescence microscopy.
Thanks and regards,
Abhijit
Hi Abhijit,
You can use THLE-3 cells which are non-cancerous immortalized hepatocytes. I have used it personally to do some experiments. They grow very slowly but they divide, so it will serve your purpose. You can get the stock from ATCC following the link: http://www.atcc.org/Search_Results.aspx?dsNav=Ntk:PrimarySearch%7cTHLE%2f-3%7c3%7c,Ny:True,Ro:0,N:1000552&searchTerms=THLE-3&redir=1.
Please let me know you have some questions.
Jyoti
If you are NIH funded, you can get PHH from liver tissue and cell distribution system at Pitt, much lower cost than from company.
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