when is the best time to split and freeze cells - confluence and passage (Mar/17/2006 )

I know it's critical to maintain your cells properly for your research. I know how to spit and freeze the cells. But very confused with when is the best time.

Is it good to always split the cells when it's 80~90% confluent? How do you determine 100% confluent? Since the cells are never seeded evenly in the flask or plates, I always have some areas that cells are complete confluent but at the same time in some other areas cells are still 70-80% confluent. Any tricks for ensure cells are evenly seeded?

Is there any standard in terms of you have to start with new frozen cells after how many passages? Say if you have 5 vials of frozen cells. You take one for routine cell culture and pass for 20 generations, then you go back to another vial. Very soon, you will run out of your cell stock. When do you freeze the cells? Isn't the later frozen cells are always several generations older than the previous frozen cells? Is that mean researchers at present time are using cells much older than the cells researcher used 10 years ago? For Hela cells, even if it is indefinite, you still need to frequently freeze the cells (not just for the sake of avoiding contamination)?

When to split cells is dependend on your cell line, but when you have large areas of your flask or plate that are 100% confluent, I'd split them. For seeding cells evenly, just shake you flask lightly and put them in a 100%horizontal incubater afterwards.

There is no standard for how many passages you can go with cell lines. Some will go for more that 150 generations, whereas others will not go over 50. So I just look at how they are performing and when they no longer do fine, I thaw a fresh batch. It's always a good idea that when you thaw your cells, after some passages you create a new and large batch of cells, that will indeed be older, but that's why I always try to make large batches in liquid nitrogen.