Leaving Cell Cultures
You’ve finally decided you’ll be taking a well deserved break from the lab and go on a vacation. The first thing you need to do after you booked your flight tickets is to plan what actions should be taken so that you can leave the lab with piece of mind. There is no use going to a vacation if you need to be checking in with your lab all day, right?
Correctly freezing cell culture
That’s it. You’re going on a vacation or you’ve just isolated a fantastic clone that expressed your gene with GFP. It’s time to treasure it (literally!) in your liquid nitrogen cell bank for future use. All cell biologists go through this tricky stage of freezing/thawing cells and there is nothing worse than coming back from a vacation to realize that a box full of frozen cloned cells could not recover from the freezing experience. Your experiment just entered the ice age at that minute! Frustrating!
Thus, it’s important to follow strict rules on how to correctly freeze your precious cell culture in a manner which will increase the chances of successfully recovering these cells.
When to Freeze Cells
- New line - Freeze the cell line as fast as possible when it first arrives at the lab and don’t forget to mark the specific passage if it is known. In case the specific passage time is not known, mark the passage time as “0” and let lab members know that this a reference point, not the “real” passage term. This is especially important to note in your lab notebook or even better, in your Labguru cell line collection module.
- Vacation outing - If freezing before going on a vacation, plan on freezing the cells a week before (or according to the cell’s expansion rate). If expansion rate is not known, find out on the ATCC collection website.
- Experiment achievement – Keep that successful stable line living for the length of your PhD.
- Primary cells – These will not live forever so you better prepare a massive stock of those.
Massive expansion – planning ahead
Cell banking is important for your future experiments so you should consider future requirements for the cells in terms of planning experiments with a healthy overhead for crisis terms (contamination, device malfunction etc). It is recommend that you generate a massive expansion of the cell source, typically 1:5 to 1:10 split onto several medium to large flasks (adapted to the specific doubling time and cell behavior). Thus, if one splits a cell line 1:5 onto five 75cm2 flasks (~7 x 106 at 100% confluence), then at 70% confluence the expansion will generate ~25 x106 cells. Assuming most cells are alive, this can account for 5 to 10 freezing vials (2-5 x106/vial).
Of course, if you plan to freeze a novel cell line, you should plan on performing differential expansion/freezing rounds every several passages to establish a large collection at different cell passages.
In my next post, I’ll cover tips for freezing and thawing your cell lines.