Growing mouse cortical/hippocampal neurospheres in DMEM:F12/B27/glutamine +FGF +EGF, and initially lots of free-floating neurospheres. After passage, neurospheres attaching to the (uncoated) 6 well plate and only a few free floating. Should I detach before passaging, or only passage the floating ones? How do I limit the attachment?
It all depends on how many passages you have gone through with these spheres. In my experience I have found that the higher the passage (or the larger the sphere) you will end up with more adherent cells. however what happenes is that sometimes those attached cells start forming spheres on their own and detach. remember the spheres is not a uniform cluster of cells. It is a mish mash of different cell types. So depending on the cells expressed they will attach or not. I would only try to passage the free floating ones for consistency. That is easy because you only take the media in the plate essentially. I would also try to avoid letting the spheres get to a too large of a size.
Hi Josef, Thanks very much. My cells are only at passage 2 though, otherwise I would have blamed that. I currently have cells from 3 independent litters. 2 out of 3 are stuck down (very few free floating) and 1 out of 3 is perfect! The frustrations of science :) I will take your advice and try to passage just the floating ones.
You didn't provide enough detail on your procedure so it is difficult to know exactly what went wrong... In theory, stem cells have unlimited capacity to divide so derived neurosphere cultures should be possible to propagate indefinitely. Neurospheres consist of 1% neural stem cells and 99% neural progenitors. However the media can vary from batch to batch and also there is a human factor involved and chances to introduce contamination. One fact about maintaining mammalian in vitro cultures regardless of the type is that usually after 10-12 passages cells become transformed. Regarding neurospheres, you have to be careful with passaging them because mechanical dissociation and vigorous pipetting can cause damage to cells. Damaged cells release stressors that effect other cells and direct them to differentiate i.e. neurospheres start to plate down because committed cells ( neuronal or glial) are adherent. When you passage cells tilt gently the flask and collect only free-floating spheres and not attached spheres. Important thing is the kind of media you used, I suggest that you use commercial media before making one from scratch. These cultures are very sensitive to even the smallest amount of detergents or impurities. They are also very sensitive to pH oscillations. Another thing is how you derived the primary cells did you combine tissues from several mice or only one. Most people use several mice to start the culture and even though these mice are inbred and should be genetically the same I've noticed that sometimes 'combined' cultures don't grow so well or differentiate faster so I believe it has to do with some immune incompatibility. Also, check your animals for any type of common viruses because we've had problems with culture when our mice got infected. One thing I recall from my work with neurospheres derived from subventricular zone is that even a type of tissue culture dish makes a difference. For example if you use 96 or 24-well plates you have a big chance that your cells will plate down. 6-well plates T-flasks for suspension culture from Sarsteadt or BD-uncoated flasks are the best to use.Stem cell are sensitive even to hydrophobic forces from walls in small wells. Even though people say that stem cells are very enduring they are in fact very gentle and quite demanding to work with.
We are not experts but we do see this happen. In the 2nd and 3rd passage, we get some settling and then as stated by Josef, they detach and form spheres. Not sure it makes a difference and I guess it depends on what you are looking at. Also, this happens more with fetal derived spheres as opposed to adult-derived spheres, but again, our experience, at present is still limited.
Hi David and Tamara
Thank you very much for your responses and suggestions. Tamara, I use commercial DMEM:F12 and supplement with B27, glutamine and pen/strep - make fresh every 2 weeks. I supplement this with EGF and FGF just before using. I don't usually pool the litters at all and plate them onto uncoated 6 well plates. Recently I have found that they don't seem to attached so much in 10cm dishes so maybe I am getting some electrostatic effects as you suggest. I'm only passaging them once before use and I'm only using the floating spheres. Having said all this, the neurospheres that I started last Thursday all seem to be attaching (beautiful spheres, but still attached!) .. :(
Will take the floating population to passage but the yield will be way down..
You can either purchase anti-adhesive plates, which are more expensive, or you can coat your cell culture plates in anti-adhesive. Previously, we used Poly 2-hydroxyethyl methacrylate. You dissolve 1 g in 50 mL 95% EtOH, warm/shake it overnight @ 37 deg C, then coat your wells on a level platform, allowing it to dry. Rinse the well once w/ PBS, then you should be ready to go.
I think it was Corning who sold them (called ultra low attachment plates?).
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