SH-SY5Y help - (Sep/21/2008 )
I am having problems growing my SH-SY5Y's and I was hoping you guys might be able to help.
I had been growing these cells in DMEM(+high glucose, l-glutamate, pyruvate)/10%FBS sucessfully for a year. These were mostly grown without any antibiotics and I had no contamination throughout this period. I went on holidays for a few weeks so I froze everything down. When I got back I found that my cells were contaminated the dreaded black vibrating dots. I tried to get rid of the contamination with pen strep and some of the other antibiotics that we have in the lab with no luck.
I went back to the lab where we obtained them from originally and found that no matter where the new lot of cells were started (3 different labs with 3 different sets of media etc) that they were contaminated with these same dots. There was no problem in the growth of the cells at this stage.
I found another lab with these cells, I got them and started up the culture. There still was this unidentified contamination, however the cells would now not grow. They would get to 30% or so confluence and then they would just detach. Because of the contamination we decided to look again, and found that lab the this guy had got his cells from. These new cells had been growing, and were not contaminated.
I get them to my lab and they look fine, growing normally (if not a little slower than the cells I was originally using), I split them which at this time they are now growing in my media. They now were acting like the last lot of cells, where they would get to 30% confluence and detach (still uncontaminated). I increase the concentration of FBS to 15% and this seemed to fix the problem, they now were growing up to 80-90%. Although they seemed to be better, there is still a problem with detachment. I split them a few days ago, checked them and they should have been ready to split today- however a lot of cells detached last night (back down to 50% confluence). I changed the media today (no wash- just get rid of the old media) and after the new media is added about half of those left detach.
When I was working with these cells initially they were very hardy and I had no problems with them at all.
Does anyone have any suggestions? Keep in mid that I am using the same media as they were originally in except for the increase in FBS, and that all were freshly opened.
I am not sure about the why your SHSY5Y cells behave this way. Just some info abt these cells. I can say that there are many variants of SHSY5Y cells around.
We have these cells which grows slowly but if it gets too confluent, they die and cells are floating. But if you would plate them at low density they also die. Also our cells start to behave different after 20-25 passages in my hands. We do not culture these till 80-90% confluency, we normally grow them till 50% confluency before splitting them and use them for experiments. Our media is the DMEM, 10% FBS, L-gln and P/S and no pyruvate.
Now a neighbouring lab got some SHSY5Y cells from else where and it behaved completely different to what we had. They had problems with their cells so they got cells from us.
Good luck !!!
Thanks scolix for your reply. I have heard of cells changing characteristics if they have been grown in different lab- it is quite a scary thought, if you really think about it. How can you really compare experiments or reproduce them if your cells are acting differently.
My new lot of cells seem to be acting a lot like your cells, they don't like to be too confluent. Although they don't really seem to mind being seeded at low density (apart from the obvious islands of cells they produce as they grow compared to a nice even distribution). I might set up a live cell imaging experiment to see how these cells grow and at what stage they seem to detach at so that I can determine the optimal time for subculturing.
I think that I just need to get used to how these cells react to certain things.
I just did a bit of extra reading on why we add pyruvate to the media, I had though (having relatively little cell culture experience) that it had something to do with providing high enery intermediates for the cells but had also thought that it was weird that you would need this as well as the glucose. In Freshney's Culture of animal cells text book, it says that pyruvate is added to increase cells ability to buffer changes in the pH of the media by generating more CO2, so it is used when no CO2 incubator is available.
I have also noticed that with these new cells, the media never turns from the fresh red colour (normal pH) to the yellow that I had seen when using the initial cells. Do you think that maybe the increased ability to buffer pH changes may cause the waste product to build up in the media causing stress and an increased propensity to detach? Keep in mind that I have more inclined to keep them in that same media for longer as it appears the right colour and also relatively clear-not too much debris from dead cells.
Every time I split our cells I add enough cells that they do not get confluent for a week and then in a week I split them. Now if the media were to change during this period this can be also kill them i.e. media turns yellow. Normally the media in our cells does not turn yellow even after a week, but if it does turn yellow, I do see much more dead cells. Our cells usually double in 2 days so its easy to split them once a week.
try to change media for your cells without splitting them. Maybe this helps. Also leave our the pyruvate and check it out. We do not add pyruvate in our media.
Thanks again scolix.
I will try to grow them without pyruvate.
When I get a chance today I'll try to post a short video of the live cell imaging I did overnight. The cells seem to want to attach to themselves not the bottom of the chamber slide.