Neurosphere Culture - (Oct/17/2013 )

I have been culturing neurospheres generated from neonatal mice.  To summarize what I've done with them...

1. Started a primary culture

2. Treated them with lentiviral shRNA

3. FACS sorted them

4. Put the sorted cells in culture (about 8,000 cells per mL.  I have them in 24 well plate)

My question is : Why aren't they growing as fast as they used to??    It's been over a week and I need to expand them so that I'll have enough cells for the subsequent assay...    How much does the seeding density affect the growth of these cells??   They don't appear to be dead and they are not differentiating either, and I'm pretty sure they are still alive, but they are just not growing as fast as they used to...   Would passaging them help them start growing faster (although I don't think i have enough cells to passage...  afraid I might lose more than I will be left with... ) ??

Any advice would be appreciated!

Thanks in advance!

Seeding density can be important, especially when there are only a few cells it's important not to seed them too thinly. As for passaging, I would wait with that till you see spheres of a reasonable size and stick to regular medium changing before. At the present time, do you see spheres (and how big are they?) or are the cells still mostly single ?

Bear in mind that both lentiviral transduction and FACS sorting put stress on the cells.

Unfortunately I don't have any other advice to make them grow faster, I'm having a similar problem myself in that I cannot expand my spheres enough for having a sufficient number of cells for subsequent assays...