Hi Joy, I haven't tried to eliminate fibroblasts but I did come across this paper which may help:"CD90 Expression on human primary cells and elimination of contaminating fibroblasts from cell cultures"

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2677147/

I am not sure of the CD status of muscle cells but it could be immune-depletion of your starting culture with i.e. CD90-Dynal beads nay give you a better chance of culturing your cells. (I have not read the paper so perhaps CD90 is not the best one to use).

Coupling beads to antibody is actually easier than it sounds (I did it quite a while ago) and generally the antibody-beads can be used 2-3x.

Sorry I can't be more help,

Gary

I used to make my own cell culture media when I needed a specialty preparation. You can also order custom media from any manufacturer, though you need to buy probably 100 L minimum. Of course, I have a pressure filtration vessel so it is easy for me to filter my media, and an ample supply of  bottles.

Can you cut back on serum? When I culture human islets, I actually use human serum albumin in the media, but minimal serum because there can be fibroblast issues if I use 10% serum.

Good luck!

Hi Joy, I found 3 companies that sell D-valine media:

https://www.funakoshi.co.jp/data/datasheet/PCI/C-75100.pdf

http://www.jazdlifesciences.com/pharmatech/company/United-States-Biological/MEM-Eagle-D-Valine-Modification-w-L-Glutamine-D-Valine-Powder.htm?supplierId=30001720&productId=492211

http://www.med.cam.ac.uk/wp-content/uploads/2012/07/paa-2011-cambs-prices1.pdf

You have to ensure to use trypsin inhibitor, elastase type 3 (15U/ml) and collagenase type 1 (165U/ml) during SMC extraction so you will be sure to reduce fibroblastic contamination.

To your SMC culture medium add EGF 0,5 ng/ml ; Basic FGF 2 ng/ml ; Insuline 5 μg/ml ; Fetal bovine serum 5%. Ensure that you medium contain Calcium also; it is essential to maintain contractility phenotype for your cells.

Hello Joy,

I was wondering if you were able to successfully culture smooth muscle cells without contaminating fibroblasts being present. I am trying to culture smooth muscle cells from mouse prostate and have not been able to find a good protocol that removes fibroblasts. Thanks for your response.