Hi,

During the first passage, it happens that confluency is not uniform. In my experience, you should split them once several colonies have reached confluency and are "big" enough (thousands of cells).

Good luck!

Hi Nese,

I would strongly suggest, you passage the cells, once you see stromal cells colonies in a good number using TrypLE. You need to be careful not to get all the adhered cells out, because with regards to mouse bone marrow, you will have an overpowering contamination of macrophages from the primary cultures when plated for the first time after isolation.

However, it is easy to get rid of them by virtue of their strong affinity to polystyrene (culture flask) in comparison to your stromal cells and they don't proliferate. You need to repeat this passaging until you get rid of the macrophage population (2-3 passages).

An alternative is to do negative selection using macrophage markers (Miltenyi kits are good) and get rid of the unwanted cells and enrich just the stromal population.

Hope this helps! 

Good luck,

Purush

I agree with Benjamin.

I isolate MSCs from patients' bone marrow. It show variation from patient to patient in the first passage. In some monolayer becomes fast from colonies, in others, I do trypsinize the expanded colonies even 70-80% monolayer is not achieved in the dish. I start with 30-40 million MNCs per 175cm2 dish obtained from Ficoll gradient method.

Subhash

Thanks for all the answers!

I also agree with Benjamin.

In my experience with BM from patients, after the first seeding, MSCs form these colonies and you cannot have a uniform confluence because of the colonies themself.

Only from the second passage you can have a uniform monolayer.

Parameters that help you to decide when cells are ready to be collected are:

- when you have a 50% of the flack covered by colonies

- the dimension of the single colony (diameter): around 0.5-1 cm diameter con be a good compromise.

- time: in my experience cells need around 12-15 days to form a good number of colonies big enough to be collected by trypsinization.

You may need a twice but very gently PBS wash to detach all the colonies.

Good luck!