Dear Researchers,
I have a problem with immortalized human astrocytes.
I did the immortalization on my own using lentivirus with hTERT (commercial). I performed the selection with puromycin and had a polyclonal line. Now, after few passages I'm observing huge amount of big vesicles produced by the cells (see attached photo) and I also suspect some cells might be dying.
I'm puzzled because I don't know if that's because I should get a monoclonal line? Or is it some cell senescence sign? I checked the cells for mycoplasm and they are clean in Hoechst and in PCR. Changing the medium bottle didn't help. I also thawed new vial and the vesicles are also there but to a lesser extent (for now...).
I am using Astrocyte Growth medium (AGM) from Cell Applications (the supplier of the primary astrocytes). During the expansion of the cells after selection I supplemented the medium with FBS and EGF but now they are on pure AGM.
Any suggestion on what is happening would be most welcome.
Kind regards,
Natalia
Dear Natalia: I will try to answer your question.
Indeed as you show in the picture your cells are entering in senescence. Compare it with the pictures in Breast cancer research and treatment 65 (2), 93-99 2001. You can also confirmed with 3H thymidin incorporation (International journal of oncology 7 (5), 1057-1060, 1995)
As described by Qu (Free Radic Biol Med. 2006 May 1;40(9):1513-23) TERT upregulation attenuates astrocyte proliferation.
As you know, immortalization and puromycin selection are big stressors for the cells. And with the recent discover, that TERT moves towar mithocondria under stress (loosing telomerase activity) (see Oncology reports 36 (5), 2731-2736, 2016), that could be part of the answer.
So far, the possible causes, let's see know possible solutions to your problem
a) Plate the cells 12 or 24 hs before immortalization with lentivirus. This will greatly improve survival of the cells following treatment.
b) Allow 3-4 days for the cells to recover before you add puromycin.
c) I do not know what concentration of puromycin you used, but try different concentrations to get the lowest that allow your selection. It varies from cell to cell, but I will start with 0.5 ug/ml.
d) Do not add ATB in the procedure medium, could lead to increased toxicity levels.
e) Starve the cells overnight (no FBS), then change the bedium with serum for 36 hr, and the apply the procedure.
f) As we mentioned before the procedure is a big stressor (due to oxidative damage), then try to add Selenoprotein-P, very useful for astrocytes. ( Free Radic Biol Med. 2006 May 1;40(9):1513-23).
g) Probably my best advice: go monoclonal.
i) I ask myself, in your study it will not be less costly and time consuming l just to buy immortalized human astrocytes?
Since I do not know many details of your research and your problem, I do not know if my recommendations were useful or not, however if you provide me more details, I will be only so happy to help.
Best,
Daniel
Dear Daniel,
I am most thankful for all your advice and I will definitely try all of it. I wasn't expected that much of help. Thank you again!
We compared the prices and it seems that even performing all my experiments on primary astrocytes would be more affordable than buying comercial hTERT line ;) That's why I wanted to give it a try.
Best regards,
Natalia
I do understand Natalia
If I can be of further help, please let me know. We do a lot of work with telomerase and the components of this holoenzyme.
Best,
Daniel
I worked on making stable cell line using puromycine.puromycin causes cell stress. And so most of the Times I ended with most of dead and stressed cells after 5-6 passages. It is better to grow cells for some time in puromycin separated with intervals of cell growth in puromycin free media. Also if u get some cell colonies, take it in a separate well and grow in selection media. I successfully made stable cells using this technique but only after 3-4 failed attempts. vesicles in cells is an indication that the cells r in stress and destined for scenescence. contrary to this geneticin is a better selection medium in terms that it does not generally cause cell stress. I got stable cells using geneticin always in one go.
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