Smooth muscle cell growth

[iym]

Hi

Does anyone here know how often you should split smooth muscle cells? I'll be seeding them in a conc. of 0.83X10(5) cells/ml (1million cells in 12 ml of media). I'd like to split once a week, in order to suit my other experiments. Is this a good starting point or do will they overgrow within a week?

Thank you,
iym

[BioWizard]

Are these primary cells? If so, you need to pay attention not to go too low with the cell density, as it might affect viability.

I think the best approach would be to look at your cells and determine if they require a minimum density to survive and divide healthily. Then, you should try to keep them somewhere between that minimum density and complete confluence.

Do you know the doubling time?

[iym]

Thanks for your reply

I don't know the doubling time. Do you? Do you know what is maximum of cells in a T75 flask?

The cells are from passage 4 though. It's human SMC

[BioWizard]

I don't know the doubling time as I've never worked with these cells. Where did you obtain them from?

How many you can grow in a T75 flask depends on the size of the cells and how fast they grow. You can look at them every day under a light microscope and split them as needed.

[iym]

I'm not allowed to be in the lab during weekends due to insurance. Therefore it is sufficient for me.

However, I read articles that the doubling time is ranging between 2-3 days for these cells. Therefore I chose to seed 2 million cells in order to obtain around 8.5 million cells after 1 week. (it is recommended to seed 2.1 million cells and they are confluent at 8.4 million cells in that kind of flask). However, after reactivating the frozen cells I saw a viability of only 1.167 million cells - hence I seeded all cell.

[BioWizard]

Yes cells usually take some time to recover from a freeze thaw. It's not a bad idea to passage everything the first time, until the cells are back up to speed. Just make sure they don't overgrow and go into starvation mode. If you're leaving them over the weekend, give them a little more media to be on the safe side.

[BioWizard]

I personally prefer not to passage adherent cells after a thaw until they reach confluence. I just change the media over them instead. Of course though no rule applies to everything in biology and it's always best to read up on your particular situation and see what other people's experience has been. If you got these cells from ATCC, you can go on their website and get lots of useful information.

[iym]

These are human cells from tissue.

I'll see how they proliferate, hopefully they have reached full confluency within a week

Thanks!

[BioWizard]

I see. Well good luck!