Dear Apoorva,

Yes, you can. I also use 10X PBS for making type I collagen gel. In the protocol provided by the company, the suggested 10X medium (or 10X buffer).

Here is the link for the protocol: http://ibidi.com/fileadmin/products/cells_reagents/R_5020X_CollagenI/IN_5020X_CollagenI_05mg.pdf

Dear Farah,

Thanks a lot for your response. Are u also developing 3D skin models using collagen-fibroblast and keratinocytes?

Apoorva

Does anyone also know the logic behind using 10X MEM or 10X PBS to collagen solution?  Is it just for dilution because for pH neutalization we are adding 1M NaOH 

pls suggest

Dear Apoorva,

adding 10x PBS may be adequate if you are preparing a layer of collagen gel onto which you subsequently seed your cells. However, if you want to make a suspension of cells within a layer of collagen, I think is preferable to use 10x MEM.  The purpose of adding 10x MEM is not to neutralize the pH of the collagen solution (which is achieved by the addition of NaOH or sodium bicarbonate), but to reconstitute a culture medium of physiological composition. If you use 10x PBS, the cells you embed in the gel will find themselves in a PBS solution, rather than in a complete culture medium. They may therefore suffer while waiting that the medium you eventually add on top of the collagen layer slowly diffuse into the gel.

I am attaching a paper which I hope you may find useful for your experiments.

Best wishes

Roberto

Sorry, I forgot to attach the paper.Here it is

Dear Roberto

Thank you so much for answering. I have come across some papers where they use 10X hanks balanced salt (HBS) with phenol red (for pH indication) instead of 10X MEM. I have HBS powder and was planing to make 10x solution out of it. 

I wanted to ask: should I supplement 10X HBS solution it  with 10 times sodium bicarbonate ? or it is to be added according to 1X??

 Phenol red I will add according to 1X HBS as higher conc. of phenol red will be toxic to the cells. Please advice 

Thanks 

Apoorva

In our hands, addition of one volume of 11,76 mg/ml bicarbonate was adequate to adjust the pH to physiological values. However you would need to check empirically if this also works with your own collagen solution (you can make a preliminary test without cells).

Good luck with your experiments!

Hi Roberto

That's a very good suggestion :) I will try a prelim exp of neutralizing collagen w/o cells.

 Just curious... do you also work with organotypic culture involving collagen-fibroblast?

Since I am presently retired, I am no longer involved in investigation, but in the past we did quite a lot of work studying the behaviour of different cell types growing in 3D matrices.

I attach a few “old” papers that may be of interest to you.

Best regards

Roberto

Dear Roberto

Many thanks to you for sharing so much information, I found the papers very helpful. I look forward to staying in touch with you.

My email ID: [email protected]

Thanks & Regards

Apoorva Verma