mesenchymal cells

always wear basic ppe and review the msdsfor safety information. dmso in cell culture freezing medium is toxic.thawing cells is a very stressful process for them to endure. to aid your cells survival,perform each step quickly and under optimal conditions, such as temperature, medium formulationand seeding density. clean and set up the cell culture hood beforeyou remove the cells from liquid nitrogen storage. you will need 10 milliliters of pre-warmedmedium and a cell culture flask. this protocol is the same for cells thawed into a suspensionflask, but the flask used will be different. be careful working with liquid nitrogen. askthe safety team at your institute if you are unsure what your ppe requirements are, orhow to safely work with liquid nitrogen.

working quickly so no cells thaw while theyare out of the liquid nitrogen storage tank, pull the vial of cells you need and placein a cup of dry ice to keep them cold in transit to the water bath.the vial of cells should be thawed in the 37c water bath quickly. be careful not tosubmerge. the vial as this increases the risk for contamination. remove the vial from thewater bath when a small chunk of ice remains inside.the ice chunk will melt as you move it to the cell culture hood. wipe the vial withethanol before placing it in the cell culture hood.working quickly, use a small pipette to transfer the contents of the vial to a 15ml centrifugetube. very carefully add 10ml pre-warmed cell

culture medium to the tube. start drop bydrop, then gradually increase your rate in order to avoid osmotic shock.spinning the cells in the centrifuge allows removal of the freezing medium that containsdmso. the speed and time may vary based on cell type.some cell lines are too delicate to spin, so this step could be skipped in certain cases.follow the instruction of your laboratory manager if your cells are very delicate.discard the medium by pouring, pipetting, or using a vacuum. be careful not to disturbthe cell pellet. re-suspend cells in appropriate volume (usually10ml) of complete pre-warmed medium. medium volume, flask size and number of flasks platedwill depend on the cell number frozen in the

vial and the optimal seeding density for thecells. sometimes a viable cell count is required in order to determine the number of flasksand media volume to be used. place the flask in the incubator with a north-south,east-west rocking motion to ensure even distribution of the cells in solution. usually cells arechecked a few hours later or the next day for good adhesion (if adherent) and propermorphology.