![]() ![]() Everything we do is outlined in the blog and here, I can’t take the credit, actually I get zero credit, this is all my team, I just write about it. So there we have it, the highest-level overview of doing iPSC culture, but all the details are here. This is a stepwise freezing process, so start at -20 degrees, then -80 after a few hours, and then the next day to their final destination, the liquid nitrogen tank where it stays until you need it again, or until someone else needs it. Based on your numbers, pipette your cell suspension into multiple cryogenic vials, and then voila, ready for the freezer. Once you have your cell pellet, resuspend in freeze down media, and calculate the cell viability and total cell number. Now for freezing cells, its pretty much the same as passaging, with one small exception, this is the end of the cells current journey as they are heading to the liquid nitrogen tank in the big storage room down the hall. You better pray someone else does, or your best bet is to simply freeze the cells down. If you don’t have a coated dish, then gulp. This plate is coated like we describe in Tuesday’s blog. These cells are pelleted, resuspended in media, and then carefully, pipetted to break the aggregates down to add onto a new plate. To do so, we use gentle cell dissociation media to lift the cells from the plates. This means removing the cells from the dish they are on, to put a small amount of these cells onto a new dish to grow again. Once the cells have reached critical mass and in our SOP, we have recommendations on when this is, then you are ready to passage. Images of what this looks like are below, monitor daily for the bad cells, when you see them try to remove them, it helps in keeping the cells all nice and pluripotent. This is like when they lose their superpowers and are no longer a superhero but just a regular person, or in stem cell terms, they lose their pluripotency. Also look out for differentiation, or when the cells become bad. But the growth rate can vary from line to line, and depends on the media you use, so be mindful of this when doing your cultures. As your cells grow, they will form tightly packed colonies that grow and expand in size over about 6-7 days until they reach confluency. There are some new approaches in which you can skips days but the preference for our group is daily media changes. ![]() How all this works is in the SOP we published on Tuesday. ![]() Also, every day you need to change the media. Your cells are attached and growing, so every day you must now keep an eye on them to make sure they are happy. Ok, enough of the fun and games, lets get down to serious business. As you can see, blue has a special place in my heart looking after my stress ball brain, he sits in on all my meetings giving constructive advice as we all work to take a bite out of the modern scientific problems of our day (haha, sorry that was an awful pun, please forgive me). But thank you for asking, they are doing well and no they don’t have their own instagram account, here is my first and last picture of my collection as they like their privacy. Before I get started, I have been inundated with requests from people asking how the dinosaurs are doing, I don’t know if they are mocking me, or if they are serious. As promised, the continuation of Tuesday’s blog in which we learn to maintain iPSCs, to split iPSCs, and yes, as it says it in the title, to freeze down iPSCs. But when its hot, what better way to cool down then with an ice cold beverage (I mean freshly frozen tube of stem cells). Man, it is a scorcher today, with a humidex of really humid, now I know how cells feel like living in an incubator. In the immortal words of Ron Burgundy “ Its so hot… milk was a bad choice”. ![]()
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