I am growing human vascular smooth muscle cells for my experiments looking at gene expression with various treatments, and have a question about procedure. Once the cells are confluent I switch them to serum free medium for the course of the experiment so they are quiescent. How long should I leave them in serum free medium before starting the experiment? They are in serum free medium for the duration of the experiment
The lack of serum growth factors affects confluent cultures of human VSMCs (and other cell types) in certain ways. Therefore, these cells can be without survival growth factors for 12-24 h. This duration includes the time frame before an experiment and the experiment duration. One can try a longer duration, however, depending upon the question in the experiment, a number of outcomes are possible. In summary, the variability is expected to increases from an experiment to the next.
I agree with Divaker Choubey. In case you want try a longer duration, then I would suggest to starve cells with very low amount of serum (0.1-0.5% FbS for example).
Hi Jerry,
As above, it will really depend on what it is you are testing. Saying that I would rarely have absolutely no serum as the cells tend not to like it. Previously we have used around 0.5% in mouse/ rat primary SMC and currently use 2% in human coronary artery smooth muscle cells. This can be lowered if you are testing peptides or growth factors that are sensitive to FBS.
The percentage we have used previously has been based in terms of what the cells are doing. In our case, we are interested in stalling the cells in cell cycle. But it is important that they can be removed from the cell stall. That is, that they can efficiently return to cell cycle when growth media/ growth factors (PDGF) or lipids (OxPLS) are applied. In this case we always determine the specific FBS concentrations to the cell line. They will survive in low (0.1-0.5%) FBS but they tend to be irreversibly stalled in our experience.
Taking all that into account it should be possible to maintain them at low serum conditions for a number of days - we usually stall for 48 hours and treat for between 24-48.
One final point about confluence is that it will be important to regulate the confluence. as the cells will continue to grow, they shouldn't be allowed to get overly confluent as they can start to differentiate irreversibly, often start to form focal point "clusters" in the wells and also shed lots of cellular materials which can all adversely affect experimental outcomes.
Best,
S
That's very helpful Scott, thanks, and thanks to the other contributors too.
Hi everyone,
anyone has experience can tell me how long the Human VSMCs take to be confluent. In my case when I culture them for the first time (take the cell's vial from liquid nitrogen), they take more than 2 weeks to be confluent, and ready to split. does this is normal?
Mine are usually confluent after a week. The time taken will depend on the seeding density and the amount of nutrients. How much serum are you using in your medium?
Hi Omar, as with Jerry ours are around a few days to a week growing in high FBS (20%) but this can be slower if they are not seeded at a high enough density. If your vials have a low cell number I would suggest initial plating in a smaller flask e.g. T25 if you are not already - In our hands SMC will always grow faster when more confluent.
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