Hi Ralph,

When working with adherent cells have to keep them on the surface of a flask at the appropriate concentration for each cell line, passing them to new flasks to prevent overgrowth. When you are going to use cells for an experiment you have to trypsinize the cells of a flask just before using them, the remaining cells you do not going to use you may pass to a new flask or a cryotube to keep them in nitrogen in case you are not going to use them for a long time.

As María says, you'll need to maintain them at the appropriate density for the cell line (this is often available online, the UK HSA being a good resource as they provide subculture routines, which gives you a starting point: https://www.phe-culturecollections.org.uk/products/celllines/generalcell/browse.jsp).

With adherent cells you will also need an appropriately treated surface, which can vary between cell lines. Some cell lines are fine on 'TC-treated flasks and plates', others will need something like gelatin or poly-l-lysine.

Check the requirements for your cell line first, or for other 'tips and tricks' (e.g. HUVECs really don't like being knocked after trypsin to try and dislodge them).

In your instance, if you had a large flask of cells (e.g. 150cm2), then I would split them 2-3 days before you need to use the first one (or on a Friday if you have a week of experiments on the Monday). I find cells intensely dislike being trypsinised two days in a row. Reseed your main flask at the recommended density (e.g. 1000000 cells in 150cm2).

Plate out the remainder at the recommended density, or above, in a number of smaller flasks (e.g. 25cm2). You may need to seed more in the flasks that will be used at the start of the week (depending on growth rate and required cell number). And keep an eye out that the cells don't over-grow by the end of the week (i.e. you may want to split your main flask again on the Tuesday to make more flasks for cells to use on thurs/fri).

Yes, it's best that you leave the cells to adhere overnight if you are plating them onto something else (i.e. 96 well, etc). So if you require Monday to Friday experiments, that will unfortunately mean a Sunday afternoon trip to the lab.

Hello Ralph Lim

Suspension cells are much easier to work with. Adherent cells need to be trypsinized whenever you have to use them. You need to plan your work whenever you use adherent cells. You can seed cells at different cell densities in a smaller flask (T-25) or larger flask (T-75) depending upon your requirements. You need to know the growth rate of your cells so that when you use those cells for your assays, they should not be confluent in the flask.

So, when you need cells early during the week, seed cells in the culture flasks at a higher density so that they are ready for use. Simultaneously, prepare flasks with cells seeded at lower density so that they get ready during the latter half of the week.

In this way, you will have adherent cells available on a day-to-day basis while the cells stay in culture. You just need to know the number of days taken by the cells at a particular seeding density to reach nearly 80% confluency in the culture flask (either T-25 or T-75). The adherent cells need to be in culture if you have to use them on a daily basis. Just to add, frequent trypsinization may be stressful to the cells.

Best.

Thanks all for the replies. I will probably passage, then divide the leftover evenly across a few smaller flasks for daily access.

An aternative possibility is to store the cells, after harvesting, in the fridge at 5-10 C.This is possible for some cell lines for brief periods ie several days.For use the cells need to be then seeded out the day before so they resume active metabolism and growth. The exact conditions for this need to be determined and optimised for your use/assay.