# How many cells should be seeded for passaging?

Do you count cells when you passage them? I found that many people will according to a ratio (maybe 1：2 or 1：3，etc.) to subpackage. It is so indistinct! What is the optimal seedling density or optimal original confluent in various sizes of cell culture dishes and flasks? that's all, thank you!

This answer is relevant in case of cell lines and not so much for primary cultures

When I first started doing cell culture, I used to count the cells before passaging. The idea is to seed enough cells to keep the population viable but not so much that you have to re-passage within 2 days. However as I became good with the technique I stopped counting as I would have a decent estimate of how much to seed. For seeding cells for some experiments (transfection/drug treatment etc) it is crucial to count the cells.

How much to seed depends on what the division time of the cell type is and how much you need after a given duration of time. You have to calculate it accordingly. For example a fully confluent well of a 6-well plate will approximately have 1 million Hela cells (this number would vary for different cell types because of the difference in sizes). Hela cells have division time of ~17h (see this post). If you need ~70-80% confluency after 17h then you should seed ~0.3 million cells.

Likewise, a T-25 flask would have ~3 million cells in full confluency. So, if you need a confluent flask in 3 days you can calculate how much cells to seed.

See this table in Nunc website for number of cells at confluency and optimal seeding density for different culture vessels.

Note that some cells (mostly primary cultures) may need to be seeded at high densities because they may need contact with other cells for survival. So, I cannot give any authoritative answer for primary cultures. Moreover, since it depends on the cell type, all examples cannot be listed here.

• I'd also argue that for special cases like transfected cells, where expression is subject to drift, you have to make a judgment call (based on experience) on whether you want to maintain or expand.
– CKM
Commented Feb 11, 2019 at 14:48

Cells should be seeded when they reach 70 percent confluence as a visual measure. Even though it is not a specific number of cells the quantitative amount of cells can be roughly measured. It is standard practice. Cells are counted with a haemocytometer they are counted and then a calculation is made for number of cells to derive a growth curve or growth rate through successive passages measured in doubling times.