Primary endothelial cells not attaching - (Jul/18/2005 )

First of all, I have very less experience in the cell culture techniques. I am working on smooth muscle cells bought from a vendor.

I cultures these smooth muscle cells till the 6th passage without any problem. After that, these cells diesn't seam to grow well. Initially I though it may be some contamination. So, started from the freeze at 2nd passage I got.

Now, When I thaw the cells, they seams to grow okay for 2 days and on the third day, I see all the cells coming off from the flask.

I also have some endothelial cells, I dont have such problem with those cells.

Can you please help me identify the source of this problem.

-Grad_Student-

HI

If the cells are not attaching, that probably means there is less or no Serum in the medium. Normally 5 - 10% serum should be ok.

-padma-

QUOTE (Grad_Student @ Jul 18 2005, 12:07 PM)

hi,
if you are observing the cell detachment after 2 days means there is something wrong in cell microenvironment. so try to feed cells with high serum(10-20%). once again check all concentrations of reagents in your medium are appropriate.
in my view, for the first time when you thaw cells it is adviceble to coat plates with human fibronectin then plate them. in this condition primary cells will adpat to artificial conditons on plasticware, from next splitting onwards you no need to do precoating of plates or flasks. this might work better.

good luck
sravan.

-payeli-

I use .1% gelatin to coat flasks - dry for ~1hour prior to adding cells, works fine. What is the recipe for the media you are using?

-bwhhms-

I use DMEM + 10% FBS + penn/strep

-Grad_Student-

Now I could grow the cells properly. There was some problem with the incubator. Although the indicator showed 5% CO2. When I measured it through external device it showed very less percentage.

Thankyou for you help.

-Grad_Student-