Technique: Freezing cells

Just this past week our lab bought two new -80°C freezers. (In contrast, a home freezer is generally -18°C). We use these freezers to store biological samples, including cells, when we’re not storing them in liquid nitrogen, a frigid -210°C.

Why do we freeze cells? Logistically, we freeze cells because we get our cells from living specimens who donate valuable cells and aren’t infinite sources. Also, although I pointed out how cells expand exponentially in my post on August 11th, they only do this near the beginning of their lives and undergo a process known as “senescence” at later age when they stop growing, unless they are true stem cells. Additionally, cell behavior and differentiation have been shown to change with continued expansion in a Petri dish, until cells might not be a good representation of their origins. Therefore, if we get a higher number of cells that we need at low passage (see the August 11th entry), we freeze them. For example, in our lab we never use our bone marrow stromal cells above passage 5 for transplants, and above passage 10 for any studies at all.

Mouse and human body temperatures are 37°C, and therefore, that’s what the incubator temperatures in our lab are always set at, to allow cells to grow at a normal rate, without causing them undue stress. As cells get colder, all of their biological processes slow down, but importantly, do not die (or proliferate). Therefore, freezing provides an excellent way of keeping cells exactly the way they are for long periods of time.

“But”, I hear you say, “surely the ice crystals that form during freezing will puncture and kill all of the cells?” That’s exactly the reason why we can’t freeze cells in normal growth medium (liquid) but instead using “freezing medium”. This typically contains 10% DMSO (dimethyl sulfoxide) which actually helps protect cells from rupturing, and is known as a “cryoprotectant”. However, we can’t use just DMSO because it is actually toxic to the cells. Therefore, the rest of the solution is generally made up of components that one would find in normal growth medium (e.g. FBS [foetal bovine serum]). There are also many different proprietary freezing mediums on the market.
The Procedure:
1) Make, or buy, freezing medium: I use 10% DMSO, 90% FBS
2) Trypsinize and centrifuge cells as if you were passaging them
3) After removing the media/trypsin solution, resuspend the cells to 1×106/mL (or as desired, but not more than 4×106/mL) in freezing medium
4) Portion the cell solution into freezing vials, 1mL per vial
5) Place vials in an isopropanol chamber (a special freezing apparatus that slows down the freezing process to avoid shocking and killing cells) and place container in a -80˚C freezer
6) The next day, transfer to a normal container box, and, if desired, transfer the cells in liquid nitrogens

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