Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

mouse bone marrow MSC culture


  • Please log in to reply
6 replies to this topic

#1 blossoms123

blossoms123

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 26 July 2011 - 05:08 AM

i am culturing the bone marrow cells of balb/c mice of 6-8 weeks. after 3 days of plating mesenchymal cells start appear(fibroblast like). But the MSCs do not multiply and after 14-15 days of culture, those cells die. I use DMEM+L-glutamine media. Please suggest me about how to get a confluent culture.

Edited by blossoms123, 26 July 2011 - 05:09 AM.


#2 Rnotk

Rnotk

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 89 posts
3
Neutral

Posted 16 August 2011 - 03:34 PM

mouse BM MSC sometimes takes long time for them to start growing after isolation, so it sounds normal for me the isolated cells are not growing right after the isolation.
But in your case, cell is dying, so first of all, I will make sure the initial seeding density, then will check the medium (especially quality of FBS)

#3 blossoms123

blossoms123

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 23 September 2011 - 01:29 AM

i am using DMEM low glucose 1x (gibco) and add Hyclone fetal bovine serum - 20%.

I flush bone marrow of 1 femur bone and seed in one 100mm dish.

please suggest what changes i need to make for cells to grow?

#4 Rnotk

Rnotk

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 89 posts
3
Neutral

Posted 29 September 2011 - 10:56 AM

you only use one femur from mouse and seeding in 100mm dish?
I think the seeding density might be too low.

Do you normally count viable cells before seeding?

Medium sounds fine to me, but also have you ever seen any contamination?

#5 blossoms123

blossoms123

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 20 May 2012 - 07:02 AM

Dear all, I could finally manage to grow the MSCs from bone marrow primary culture .

But, i face a new problem.
m unable to detach all cells from a T25 flask by Trypsin EDTA. I keep the trypsin at 37degree b4 using. I wash the cells twice with TPVG and add 2ml more TPVG and keep for 2-3 min at 37 degree but still all cells don't detach.
Due to repeated TPVG exposure cells are also getting stressed.


Please pls.. help.

thanks.

blossoms123.

#6 Tabaluga

Tabaluga

    Making glass out of shards

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 401 posts
49
Excellent

Posted 20 May 2012 - 08:09 AM

Have you already tried giving the sides of the flask some slaps with your palm after trypsin exposure ? This mechanical influence can help (at least it did for me)

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#7 Open Biotechnology

Open Biotechnology

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 17 September 2012 - 10:28 PM

You could try incubating with trypsin longer. I've had trypsin go bad on me before as well.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.