Hi,
I'm trying to make an adult (!) primary astrocyte culture from mouse cortex. I am doing this with a very experienced person who has done this before in a different lab. I am using her reagents and protocol, and at the last stage before plating we are using a viability assay and automatic cell counter, we have a lot of alive cells (around 70%) and then we are plating them at the density suggested by her protocol. All of this seems to be going well.
But then, if I check 4h or 24h later, none of the cells attach. Sometime I see very small cells attaching which I guess are bacteria (looks like it). Here are the things I tried: Poly-L-Lysine coated cell culture flasks (the "normal" T25 flasks), Poly-L-Lysine coated dishes, Fibronectin coated flasks, uncoated flasks, Poly-L-Lysine coated glass coverslips, Poly-L-Lysine and Laminin coated glass coverslips.
Nothing attached to any of these surfaces! I also tried different media that were tried and tested and used in other labs (Astrocyte Basal Medium and DMEM-based recipes), but I think the problem is rather in the attaching.
Other primary cultures in my lab are doing fine on the PLL coated surfaces, so there doesn't seem to be a problem with the coating itself (I read PLL can be toxic to neurones if not done properly).
We are all out of ideas.
I know I haven't given details about my culturing protocol, I think that is fine, so did anyone ever have an overcome this problem with attaching? Or do you think it can be due to the medium after all?
Cheers!
I faced the same problem during primary cell culture, though i was working with epithelial cells. For attachment of cells, I plated my cells in media containing 10% FBS and changed the media after 3 hrs to serum free one.
Not sure whether it will work in your case or not
Hello,
I culture astrocytes often and never have had a problem with their attachment. I usually do not use Poly-L lysine, except for glass coverslips and Human astrocytes. Maybe the issue is with your media? I use: DMEM with 10% FCS+ 1%NEAA+ 2mM glutamine+ pen/strep.
You should be good to go!
Hei Laura,
I had a similar problem with adult rat astrocytes and I used Matrigel Matrix for attachment. It worked quite ok, but not for each isolation. Also, for you media, try to use at least 10% FBS; I tried with 15% and it worked much better.
Hope you suceed!
Hi Laura, I work on primary glioma cells and we also use 15% FCS, I would even do some more at least for the time of adhesion/(you can still work on them at lower concentrations). Anyway another idea would be to coat your flasks with Fibronectin. It happens that astrocytes really rely on such a matrix, if they do not produce it on their own.
Another suggestion would be to take the cells first into spherical culture (FCS free medium supplemented with growth factors) and then try to grow these spheres with Serum and see if they attach
Hope it will work
Laura, given that the protocol you are using has worked before I don't think changes to the protocol (e.g., medium formulation, plastic coating, etc) are necessary. In fact, I have found that astrocytes grow pretty well in DMEM with 10% FBS, I suspect that small deviations in this formulation will make little difference - though I should state that I was growing primary astrocytes from 3do mouse pups.
In any case, I think you are at the stage where you may need to start anew. Remake everything and check that each component is prepared exactly as it should be. Perhaps let the person who is assisting you perform the entire procedure and take note of everything, down to the finest detail. Protocols often miss these finer details.
Also, re-consider the plastics that you are using, we had an issue with flasks that were okay for cell lines, but not okay for our primary cells. If possible, use the exact same plastics, sourced from the same company as the person who has performed this protocol successfully.
This is my default troubleshooting approach for protocols that have worked in the past and have suddenly stopped working. Better this than going through the arduous task of tweaking your conditions for what may well have been fixed by something as simple as replacing the plastics.
And your statement "I see very small cells attaching which I guess are bacteria" is a bit concerning and the presence of bacteria in your cultures certainly wouldn't help with growing primary cells. Though, can't say that in the rare instances I have had bacterial contamination of my cultures have I noticed bacteria attaching to the plastic.
Also, make certain that your solutions do not contain any sodium azide. If you are selecting astrocytes by a using an antibody (usually conjugated to pull the cells out of the mixture) be certain you are purchasing the sodium azide free version. Your cells may pass the cell viability assay, but will not survive beyond that.
Okay, I'm out of ideas. Hope these will be helpful.
Best of luck.
A lot of good advice proposed here already.
I agree with Ricardo that you definitely need to rule out bacteria. Also you should test them for mycoplasma infection as this can affect attachment, proliferation and viabilty after thaw (with out obvious signs of infection).
I've been culturing lots of astrocytes lately and never had any problem with attachment (I use Poly-ornithinin ~1-5ug/ml). The only occasion I coat with laminin is after thawing astrocytes or glial progenitors from cryostorage.
Astrocytes produce their own laminin and thus should not need additional coatings for attachment. With this in mind, astrocytes do have a tendency to flatten and flow out if seeded at low confluency and without coating.
Good luck!
OT: I link a paper including a small comparison of different coatings, note that they look at survival rates of neurons, but perhaps could be of use if you want to experiment.
http://www.nature.com/ncomms/2014/140313/ncomms4449/full/ncomms4449.html
Dear Marco,
sorry for the late reply I just saw your question.
In the end I found a solution but it's a lot of effort: I make a culture of newborn astrocytes, this is very easy and doesn't need anything fancy, it should work for you.
Then, I use the medium from these astrocytes ("conditioned medium") to extract and plate astrocytes from the old mice. That means, I put fresh medium on the astrocytes for a few days, and then mix this "old" medium with new medium 1:1 and use that. The rationale is that the astrocytes produce their own, astrocyte friendly, environment and I am using this to culture new ones, so they are happy from the start and get stimulated to grow.
I still don't get loads of astrocytes but it seems to work (I'm not doing this experiment anymore at the moment). This way, you always have to have a culture of astrocytes going to produce the conditioned medium if you want to cuture new cells.
I hope this helps!
Laura
Edit: and also, patience. It happened to me that I couldn't see any cells after a few days, but once the astrocytes start finding each other they start to grow faster. You will find patches of them that can be easy to miss.
i usually do a lot of astrocyte culture... i do not face that problem . i use PDL (poly- D- lysine) coating. i would like to share my protocol for your convenience
hi all!!
i am trying to culture adult human brain cortical cells from autopsy brain tissue. but my culture do not adhere or grow. kindly help regarding the same .
The brain sample were taken within 12 hours of post mortem time. this the procedure i followed: firstly the plates were coated with poly d lysine . the plating media used was dmemf12+10 Fbs+ NEAA+ bFGF+ pen strep+ gentamycin. i used a 2 min treatment with 0.1 % trypsin to dissociate the cell. and further titurate using a 1ml pipete tip.
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