Hi, I am doing a project about hemodynamics on smooth muscle cells.
I tried to embed cells in 3mg/ml collagen, but most of them died 1d later.
Here are details, would anyone help me to find out what I am doing wrong?
1 According on product protocol, 5mg/ml collagen mix with HEPES and 7.5%NaHCO3 on ice.
2 harvest cells and mix cells with collagen,final concentration is 5*10^5 for cells, 3mg/ml for collagen
3 incubate the mix for 30min
4 add medium
5 cell dies
I also tried EC, died again
Hello. If none of the required components is missing and all recommended steps were carried out it could only be inproper pretreatment of cells or the mixing precedure is too harsh. I would recommend doing some control experiments in parallel to systematically rule out what is harmful to the cells. Do the cells have access to nutrients and oxygen, is the hydrogel layer propably too thick for cells getting nourished by passive diffusion ? Best luck
In comparable experiments I did, I had incorporated 10x medium before gelation started, so cells were sourrounded by medium already during gelation / fibrillogenesis, not only afterwards ... maybe send me the link to your product manual, then I can give further advice.
Martin Vielreicher Hi Martin, thanks for your suggestion! Sorry for the late reply!
Yes, you are totally right, I use 10XM199 for my smooth muscle cells before gelation, I also modify my pH, now cells are really perfect!
The thickness of the collagen layer is really a big problem, I reduce the concentration of the collagen, cells are much better now!