3D collagen matrice - Polymerization of collagen gel (Nov/09/2006 )
Hi, this is my first time here...I'm trying to do 3D collagen matrice. is there someone who is experimented in this field? I'll explain troubles encountered after.
Thanks
Thanks
I work with 3D collagen type I matrices. What's your trouble?
Hi,
I will first gave you my protocol : I used Purecol (vitrogen, 3mg/ml stock), 2X MCDB 202 medium (NaHCO3 added) and Hepes.
I first tried 1.25mg/ml for collagen final concentration (4parts of collagen), 5parts of medium with cells (fibroblasts) and 1 part Hepes 0.2M. Put 10ml of this solution per 60mm bacteriological dish. The gel polymerization took around 2h! Seems to be long and the gel were very not very strong...For detachment from the well, it supposed to be easy just by adding medium once it's polymerized but it's not always so easy! I sometimes need to used a spatula for detachment...probably endommaged gel! I also tried to put 1ml per 24-well not very better...
Then, I decided to tried 2.4mg/ml as collagene final concentration : 8part Collagen, 1part MCDB 10X and 1 part Hepes 1M. My gel were not polymerized at all!!! 
The pH of the final solution is between 7-8.
I hope you'll be able to give me some help! 
Thanks
For the collagen solution a 1.25mg/ml, I'm not astonished that the gel is not very strong. Sometimes I use collagen at 1.5mg/ml and the gel is not strong (but I don't need to remove it so I don't mind). I'm not astonished either that it takes 2h to polymerize if you use a volume of 10ml. How big is the volume how long is the time took to polymerize.
Concerning the detachment from the well, it seems to me it's hardazous to do it with a 60mm diameter gel. Why do you need to do it? Do you remove the gel from the dish?
My collagen solution is a little bit different from your.
I use 2mg/ml collagen in acetic acid solution, 10x DMEM, NaHCO3 and NaOH (Is your collagen stock in acetic acid solution? I yes, do you equilibrate the solution with NaOH? I guess yes because the pH is between 7 and 8...)
Final solution is: 7,5 parts of collagen, 1 part of 10x DMEM and 1,5 part of NaHCO3 15.6mg/ml.
I sometimes use another protocol to make gels on which I culture cells, but the only differences are that I use MEM and 10x HBSS in place of DMEM, I add FBS and the collagen concentration is higher.
I hope this may help. Don't hesitate to ask questions if I'm not clear enough.
Concerning the detachment from the well, it seems to me it's hardazous to do it with a 60mm diameter gel. Why do you need to do it? Do you remove the gel from the dish?
My collagen solution is a little bit different from your.
I use 2mg/ml collagen in acetic acid solution, 10x DMEM, NaHCO3 and NaOH (Is your collagen stock in acetic acid solution? I yes, do you equilibrate the solution with NaOH? I guess yes because the pH is between 7 and 8...)
Final solution is: 7,5 parts of collagen, 1 part of 10x DMEM and 1,5 part of NaHCO3 15.6mg/ml.
I sometimes use another protocol to make gels on which I culture cells, but the only differences are that I use MEM and 10x HBSS in place of DMEM, I add FBS and the collagen concentration is higher.
I hope this may help. Don't hesitate to ask questions if I'm not clear enough.
Others questions:
Finally at 2.5mg/ml, my gel took all night to polymerised do you think it's ok for further experiments using cells...to be in gel without any medium for around 16h? (I tried it before without cells)
I don't mind either to remove the gel from the dish, what I want is to evaluate contraction of it! So I now made my gel in 24-well plate (1ml per well)
I didn't need to adjust pH with NaOH, with the solution I used it ended at pH between 7-8...The NaHCO3 (1.7g/L) is added in the medium MCDB. Do you think I should change my mix?
Finally, how did you detached your gel from the dish? Maybe you don't need to, depends on what you are looking for...
Thanks a lot for you help!
Doris,
I answered you by e-mail...