neurospheres - (Sep/15/2006 )
hi all
has somebody worked with mouse/rat neurospheres? We bought ones but they didn´t work, didn´t last more than a week. Can you maintain them without differentiating for 3-4 passages? how experiments could I do with one vial (which is expected to contain one million of neurospheres)?
thanks in advance
has somebody worked with mouse/rat neurospheres? We bought ones but they didn´t work, didn´t last more than a week. Can you maintain them without differentiating for 3-4 passages? how experiments could I do with one vial (which is expected to contain one million of neurospheres)?
thanks in advance
You should be able to keep neurospheres in culture much longer then 3-4 passages. Who did you buy them from? Do you keep them as suspension culture? What media? You don't have to differentiate them, but to maintain viability you should dissociate them to single cell suspension once in a while. That way cells generate secondary spheres and you end up with lots of them. Also, proliferation rate is concentration dependent...
Thanks.
I bought them from StemCell. I must say that I was wrong, the vial has 5 million cells.
They give you the vial from which you get neurospheres who are expected to float and grow in flasks. Then you have to subculture 6 - 8 days after plating (first dissociating them to single cell suspension), and a week after this, if you want to differentiate them into neurons, astrocytes and oligodendrocytes, you have to plate them in culture dishes with Poly-L-Ornithine and use the "differentiation medium". All the medium I used was from StemCell, (Neurocult media).
My neurospheres died 2 weeks after first plating, I never reach the step of differentiating. As they are very expensive, what I like to know if I can maintain them undifferentiated passaging them as usually with other cultures and then when I want to experiment just take them and differentiate them in the coated dishes. I hope it is not neccesary to buy a vial every time you have to experiment!
thanks again
I bought them from StemCell. I must say that I was wrong, the vial has 5 million cells.
They give you the vial from which you get neurospheres who are expected to float and grow in flasks. Then you have to subculture 6 - 8 days after plating (first dissociating them to single cell suspension), and a week after this, if you want to differentiate them into neurons, astrocytes and oligodendrocytes, you have to plate them in culture dishes with Poly-L-Ornithine and use the "differentiation medium". All the medium I used was from StemCell, (Neurocult media).
My neurospheres died 2 weeks after first plating, I never reach the step of differentiating. As they are very expensive, what I like to know if I can maintain them undifferentiated passaging them as usually with other cultures and then when I want to experiment just take them and differentiate them in the coated dishes. I hope it is not neccesary to buy a vial every time you have to experiment!
thanks again
Yes you can expand them as suspension culture and you can even freeze them so that you can have extra stocks. I have never used the cells from stem cell tech. (we isolated them ourselves) but I used their kit for chemical dissociation and that is the only method that really gave me single cell suspension. And my impression is that all the products they have are good. If you want to expand the culture, plate the cells at higher density (more than million per ml) and then monitor so that you can feed them on time. Do not let the spheres grow to big, i.e. you do not want to see the dark center and the cells dying at the surface. So by passaging (dissociating) them more often in the begining you can generate more cells and have enough for freezing and for exp. I noticed that they grow faster if plated in 6-well plate.
thanks a lot 
I also think that products from stemcell are good, we often use them in our lab and they work very fine.
Is it difficult to isolate them from mice/rats? Are they very fragile? I mean, I´m not still very used to work with animals and brains but I would like to isolate them myself if I could, because they are quite expensive.
I also think that products from stemcell are good, we often use them in our lab and they work very fine.
Is it difficult to isolate them from mice/rats? Are they very fragile? I mean, I´m not still very used to work with animals and brains but I would like to isolate them myself if I could, because they are quite expensive.
No, it is not difficult. We did it with postnatal day 1 pups...check your inbox messages
Hello,
I work for StemCell Technologies and I am very concerned that you were not able to get our neurospheres to grow successfully. If you are still having problems, please feel free to send me your email address and I will have our technical support department contact you (or you can email them at info@stemcell.com). We would really like to be able to work with you to get your neurospheres to grow successfully.
I do have a few initial questions, which might help pinpoint the problem:
1. How long did you leave the thawed neurospheres before disaggregating and passaging?
2. Did you add cytokines to the medium?
3. Did the cells die or did they attach to the flasks?
4. Were you able to passage at least once? What was the viability after passaging?
We will also want to know the lot# of the cells and the medium you used, if you still have it so we can ensure we have complete records of our products.
I look forward to hearing from you.
Diane Miller
Product Manager - Specialized Media Products
StemCell Technologies