Help! neural stem cells culture - no neurosphere after P1 - Please troubleshoot my protocol (Jan/23/2007 )
Dear all
I am starting culturing neural stem cell from mice hippocampus, but I find it is much more difficult than what I expected. The problem is I cannot obtain neurospheres after the first passage. I think this is due to some steps in my culturing protocol, but I am not sure. I am glad if you guys can give suggestions.
Neural stem cell (NSC) culture started (day 0)
1) isolate hippocampus from E14.5 mice embryos (1 mother gives averagely 13 embryos) and put in Advanced DMEM/F12 medium until all the embryos are dissected (normally within 40 min)
2) wash the hippocampus with PBS
3) add 3 ml 0.05% trypsin (with EDTA), pipette up and down with a 5 ml pipette, incubate in 37oC water bath for 10 min
4) neutralize the trypsin with 3 ml Advanced DMEM/F12 medium supplemented with 2% FBS
5) repeat steps 3 and 4 for 2 more times (some sticky, glue-like hippocampus remained)
6) pass the medium (but not the glue-like substance) through a 40 um cell strainer to remove clumps
7) wash the cell strainer with 10 ml PBS trice
8) centrifuge the cell filtrate at 1400 rpm for 5 mins
9) resuspend the pellet in culture medium (Advanced DMEM/F12 medium supplemented with 20 ng/ml bFGF and 20 ng/ml EGF), perform cell count and viability test (trypan blue exclusion assay), viability mostly = 85-90%
10) adjust cell density to 1E6 viable cells by culture medium (Advanced DMEM/F12 medium supplemented with 20 ng/ml bFGF and 20 ng/ml EGF) and 20 ng/ml EGF and culture in 75 cm2 tissue culture flask, incubate at 37oC, 5% CO2 for 4 days (the cells are mostly discrete single cells)
Change medium (Day 4)
11) change medium at day 4 (many floating spheres are formed with about 40 um in diameter, no spheres and single cells are adhesive), the medium containing the spheres is centrifuged at 800 rpm for 5 mins, resuspended gently in culture medium (Advanced DMEM/F12 medium supplemented with 20 ng/ml bFGF and 20 ng/ml EGF) and culture in a new tissue culture flask, incubate at 37oC, 5% CO2 for 4 days
Passage 1 (Day 8)
12) passage (P1) at day 8 (many large floating spheres are formed with about 100 um in diameter, a few spheres and cells are adhesive), the medium containing the spheres is centrifuged at 400 rpm for 5 min, the supernatant is discarded
13) resuspend the pellet in 3 ml 0.05% trypsin (with EDTA), pipette up and down with a 5 ml pipette, incubate in 37oC water bath for 10 min (a glue-like substance appeared again)
14) pipette up and down with a 2 ml pipette, neutralize the trypsin with 3 ml Advanced DMEM/F12 medium supplemented with 2% FBS
15) transfer the medium (but not the glue-like substance) to a new falcon (the medium is quite clear). Centrifuge at 800 rpm for 5 min, discard supernatant
16) resuspend the pellet in culture medium (Advanced DMEM/F12 medium supplemented with 20 ng/ml bFGF and 20 ng/ml EGF), perform cell count and viability test (trypan blue exclusion assay), viability mostly = 85-90%
17) adjust cell density to 5E5 viable cells by culture medium (Advanced DMEM/F12 medium supplemented with 20 ng/ml bFGF and 20 ng/ml EGF) and 20 ng/ml EGF and culture in 75 cm2 tissue culture flask, incubate at 37oC, 5% CO2 for 4 days (the cells are mostly discrete single cells)
18) after 4 days (Day 12), for spheres, only 1-2 large dark brown spheres are floating and several ‘healthy’ one are adhesive on the bottom. For single cells, many cells are adhesive but still, many are floating.
So, my questions are:
1) is the glue-like substance in step 5 normally present? As it is the undigested or incompletely digested part of hippocampus, will this affect the extraction of NSC which is mostly resided in dentate gyrus?
2) If yes in question 1, what are the spheres formed in day 4? Are they just cell aggregate of single cells? Or truly neurospheres derived from progenitor cells or NSC?
3) What is the most suitable cell density when seeding cells in step 10?
4) Why those single cells in step 11 (day 4) do not adherent to the bottom, as they are supposed to be neural cells? And why after 1 passage, they are adhesive (Step 18)?
5) For the tissue culture medium, I do not include N2 or B27 supplement, is it still ok for NSC growth?
6) Is it ok to use trypsin to dissociate NSC cells from neurosphere during passage? What will the glue-like substance be in step 13 after trypsinization? Will this substance account for the low formation of neurospheres after passage 1?
7) I have read many papers using ‘fire-polished pippete’ to ‘mechanically dissociate’ the neurospheres. What actually is the ‘fire-polished pippete’? how to fire-polish them? And is mechanically dissociation equals to triturating the neurosphere? Can I use a 16G or 18G needle instead?
Since I am the only one in our Department started to do NSC research, I do not have much opportunity to discuss NSC issues with others. So I will be very happy if you guys can help me~ Thanks x 1E6!!!
hi,
actually. use the polished pasteur pipette will reduce the viability of the neurospheres.
your research is really clost to my research. I also use the syringe needle to do the mechanically dissociation. besides, u also can use pipette tips.
ok, good luck.
to make sure that the spheres that obtain is really neurospheres , better u stain the spheres. use nestin.
Hello,
It seems like you are having multiple problems, which may be difficult to sort out all at once. I do have a few comments which might be of use.
1) The glue-like substance is most likely DNA from dead cells.
2) Using FBS with neurospheres encourages differentiation of the cells which means they will adhere to the flask and not passage as neurospheres
3) We have never tried Advanced DMEM, but for neurospheres to grow they do need an appropriate supplement added (like N2 or B27 or our NeuroCult NSC Proliferation Supplements).
4) Many researchers believe there are no neural stem cells in the hippocampus. If this is true, it explains why you cannot passage your cells.
We do offer a great technical manual for the culture of mouse neurospheres. There might be some information in this manual that you find useful including recommended cell density, etc. It is free to download off our website: http://www.stemcell.com/technical/28704_neurocult.pdf
Feel free to contact me, as I would be happy to offer more suggestions.
Diane Miller
Product Manager - Specialized Media Products
StemCell Technologies