The Half Decent Pharmaceutical Chemistry Blog

I must admit, I haven’t seen the film mentioned in the title of the post, but I guess that’s something I share with many of you. We all know what it’s all about: a skinny, red-haired, German, inexpressive girl, who runs for all the damn 81 minutes the film lasts, trying to find the money to save her boyfriend, everything garnished with techno soundtrack. How disgusting is that?! Frankly, only a completely untalented director could have come up with such a stupid idea. However, in the video-clip era, some useless critic might have been crazy about such a pointless thing.

Annoyingly, this film popped into my head this morning, when I was to conclude my daily session in the tissue-culture lab. Perhaps that was a consequence of the appalling headache I was beginning to suffer (and, partly, it’s not completely gone yet). This was, indeed, a nice reward for my committed work: as said last week, I’m to assay cytotoxicity and to get this job done (quite) precisely and quickly, I’m going to employ MTT. So, plainly, I was plating out some cell-lines, as I’m first about to determine the ideal number of cells for each line. This involved preparing lots of different dilutions and some counting with…,well, the counting chamber. Thing is, that lab is nicknamed “The Fireball”: there wouldn’t even be need for an incubator there but you could perfectly leave your flasks on a table and cells would grow normally.
So: a very hot room, a long work and headache. How fair is that?

Still, I finished with approximately 2 millions of cells to be stored in a so-called cryotube to, as you might expect, cryopreserve them. And this brings back the odd image of that hopeless woman running. Let me explain. Once you’ve neatly concentrated your cells in 1.25 mL of cryo-medium (argh!), you have to place the test tube in the -80°C freezer (before eventually storing them in liquid nitrogen) as quickly as possible. Even when you suspend the cells you can’t waste any time: in fact, after a 5 minutes 1,100 rpm centrifugation (at room temperature), which yields a lovely pallet of cells, you discard the supernatant very quickly (it still contains a bit of the trypsin you used to detach them from the flask and, at this point, you want keep all of them as a pallet) and suspend them again in a small amount of cryo-medium before you put everything in a test-tube.

There are different “recipes” for cryo-media, basically function to the type of cells. For today’s primary fibroblast, the chef, err…my supervisor told me to use a pretty standard 50% FCS, 45% DMEM and 5% DMSO (if you don’t know what the first two abbreviations stand for, shame on you). Yes, there’s a bit of chemistry in this post! DMSO is always added and it’s a fundamental ingredient being a cryoprotectant: if not present, cells will inevitably be damaged by the formation of ice crystals.
With the cells in the tube, to do things by the book you should allow them to cool down slowly but constantly, before storing them at -80°C or whatever. There is a variety of tools to complete this task but we don’t have any of them and, as far as I can tell you, although we’ve been recently awarded a mind-blowing 40,000 € fund from a private foundation, as far as I can tell you, we’ re not going to spend a cent on such a device either. So, what we do, instead, is placing the tube in the fridge the Fireball is featured with. When it’s cold enough, you put the cryotube in the freezer, which is in a separated building: so, it’s not unusual to have, say, a run in the park between the two buildings, generally wearing a shirt because the lab coat must remain there to avoid contaminations and you’re not in the mood of wearing anything more because you are very hot. However, you have to be fast not to shock the cells with the different temperatures (from +4°C in the fridge, to +38°C in the Fireball, to whatever is outside, to -80°C).

Run, baby. Run.