When maturating neural progenitors cells to mature neurones we use a modified protocol of 30 days of maturation from Jiao et al., 2013, Hum. Mol. Gen.
We coat the wells with 0.5mg of Corning Matrigel for every 6 wells of a 6 well plate.
To start the maturation, we passage the NPCs (in 6 well plates) and seed them on Matrigel-coated coverslips in a 24 well plate (we do not use astrocytes, only neuronal culture). We wait until the confluence is around 80% and we change the media to the maturation media specified in the paper above mentioned (usually takes 3-4 days).
The problem comes around the DIV 21. When changing media (even being extremely careful) cells detach easily. Even when moving the plate, the shaking it produces, makes the cells detach.
Has anyone experienced this? Any ideas on how to overcome this problem?
Have you considered plating you NPCs instead of matrigel on Poly-L-Ornithine and Laminin? Matrigel is not very stable after two weeks. For long term culture you can add Laminin into your culture medium.
Hello Kristine, thanks for your answer. We thought exactly the same!
We're currently trying with PLO/laminin and adding laminin after 3 weeks of maturation in long-term culture with Matrigel coating. We have to see how it goes.
The thing is that when we try to find other people with the same problem, it seems that we are the only ones experiencing this (even people using Matrigel say they do not have this problem) and it is very difficult for me to believe that we are...
Thus, I am asking to see if other people are experiencing this and if there are any other solutions.
I would also suspect that the problem might be the coating. Matrigel is not stable that long and you might want to give it a try with coating with Laminin, etc. as Kristine suggested.
Hi Pedro,
There is a neurodevelopmental explanation why NPCs are stickier than neurons, but we can discuss it another time ;) However you can force them to stay on site in different ways as correctly suggested by Ceren and Kristine. I would also like you to consider some other issues, such as the correlation between the volume of media, homogenous pressure on top of the cell culture and meniscus effect. Coverslips' pre-treament can also influence the attachment. Etc Etc. I also recommend you to check "The Corning Guide to Surface Selection by cell type", which offer a nice overview about coatings.
Cheers,
Marco
Don't worry, we had the exact same problem, so you are not alone.
The neurons looked fine in the beginning and started detaching after a while. We also used Matrigel and recently switched to a Poly-D-Lysine/Laminin coating. No problems after that. Matrigel is fine for short-term cultivation, but it is unsuitable for long-term cultivation.
Good luck with your cells!
Ceren, thanks for your answer! Thanks Thomas for letting me know! We plan to use Poly-D-Lysine/Laminin coating also, good to know that it worked for you.
And Marco, as always, thanks for your help! We can talk about all these issues you suggested soon.