So, I want to extract bone marrow from mice for an experiment, but I don't need all tens of millions of cells that I would get. So I was thinking that I could freeze down the left over cells for future use. I would want to eventually culture the cells into eosinophils with SCF/Flt-3 and IL-5 media. But I've never frozen down freshly extracted bone marrow (or anything, actually) and neither has anyone in my lab. What would be the best procedure for this?
Hello Cassandra Poole
Yes, you can.
Fresh bone marrow cells may be counted using the hemocytometer, and then centrifuged for 5mins, at 200 x g at 4 ºC, and gently resuspended in freezing media containing 90% FBS and 10% DMSO. You may freeze 4-6 x 10^6 cells/ml/cryovial by gradual freezing technique at-1ºC/minute maintained in a -80ºC freezer for 24 hours and later transferred to a liquid nitrogen tank.
Later, when you need these cells, you may thaw the cells by a quick-thaw process wherein you quickly transfer the cryovial containing the cells to a 37 ºC water bath until just the tiniest ice crystal is left in the cryovial which usually takes 1-2 mins. Do not vortex the cells. After thawing the vial, immediately transfer the cell suspension to a large volume of pre-warmed growth medium (~10 mL/1-mL aliquot of cells). Centrifuge the tube at 700rpm for 5 mins, discard the medium, resuspend the thawed cells in fresh culture medium. Culture the cells in an appropriate culture vessel.
Best.
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