VSMC culturing - (Aug/17/2007 )

Help! I've just started using rat vascular smooth muscle cells and the protocol for general culturing/passaging doesn't seem to be working. I have so much trouble getting the cells to detach, and when I finally get sufficient numbers (~50%) to detach, they look so sick and deathly!

OK, so here's what I'm doing: pre-warmed PBS/EDTA: 1 wash, then another 5 min wash of 3mL (cells in incubator); 1 mL trypsin 5-10 min (cells in incubator), hit the flask a couple of times, and if I'm lucky, I may get 30-50% cells detaching. What am I doing wrong??

Thanks!
Z

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Dear Zoe8,

There are plenty of things to try. I have to say that our VSMC cells come off pretty well. There is never a standard way of doing things, all parameters have to be OPTIMISED. Here goes:

1). Always passage/subculture your cells PRIOR to full confluency i.e. at 70/80%.

2). If your cells attach too well to the plastic you are using TRY another manufacturer....each TC plastic has differing properties.

3) Wash your cells x 3 with PBS. This is done in the cabinet at room temperature. The cells should be in contact with the monolayer only for 15 -20 seconds. The whole idea of washing is to reduce/dilute the Ca/Mg concentration and the protein concentration from the media......this is what normally determines the efficiency of the trypsin.

4). The trypsinisation should be done at room Temperature and if the washing has done it's job then the cells should detach within 30seconds-1 minute. 5-10 minutes is far far far too long. Check the concentration and age of your trypsin.......0.05% is normal. You can buy more concentrated Trypsin solutions, it maybe worth titrating a solution which detaches the monolayer, but does not kill the cells. What is the size of your flask:
T25cm flask.........1-2mls of trypsin/Versene
T75cm flask.........3-6mls " " "
T175cm flask.......6-12mls " " "

5). Are these primary or cell lines. If primaries are they PURE VSMC or have you got fibroblast contamination?

Hope this is useful

Rhombus

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Hi Rhombus,

Thanks for your help. I'll give your suggestions a try. I find it hard to judge the confluency as they appear to grow on top of each other as well as across the flask. I've been using 2 brands of tissue culture plastic but the cells stick well to both types.

The cells I have are primary and their purity was determined by the girl I got the cells from & supposedly fibroblast-free.

Thanks again!

Zoe8

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