Cell Culture: Preparing mammalian cells for storage

I have been working in vitro with monocyte cells for the past few months. I was fortunate to receive a flask of already cultured cells from a neighboring lab – fortunate because my lab did not have to pay to procure them. However, that meant I did not have a stock supply or a back-up  of the cell-line. Once you start working with cells, they have to be maintained at least every 2 to 3 days. There are only two of us currently working in my lab, so that means if I am away from the lab for an extended period of time I will have to freeze down aliquots of the cells for storage. This should be done with every cell-line you work with for the sake of continuity, amongst other reasons. Cell-lines age as you continue to work with them, and in time may begin to change in characteristics.

The process is slow but simple. Below is a brief description of how it’s done:

First, collect cells – suspended in medium – in a tube.Taking a small sample of the cells; “trypan-blue” is added to help count the number of live cells. Dead cells will take up the blue dye; do not count these!

hemocytometer, trypan blue and, manual counter

Place a small amount of trypan-blue cell suspension onto the hemocytometer (the rectangular glass object). This device has grid-lines that aid in accurately counting the number of cells. View under a microscope like this: cellcounting4

Once you know how many cells you have, you can determine how to distribute them. Aliquots (equal portions of the whole) are placed in cryo-vials. Cells are suspended in “freezing medium” that keeps them from bursting when frozen, as well as, supplies nutrients for them to stay alive during storage.

Aliquots of cells in cryo-vials; freezer box.

Once you have all you vials filled, label them with the cell-line name, the date, the number of cells, and your initials. The cells are then placed in a freezer storage box, standing upright, and place at increasingly colder temperatures overtime. Finally they can be stored at -80 degrees Celsius for short-term storage, or in a liquid nitrogen chamber, for long-term storage.

That’s all folks! When I get back I will thaw a sample and get right back to work. Until then, I will be absorbing some U.V. rays in sunny Barbados!

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