We recently started working with human osteoblast cell line SAOS2. We froze the cell line in 15% Mc Coy's 5A medium (1 ml) with 5% DMSO for cryo preservation. after trypsinizing and adding the DMSO, we let them stay in an isopropanol box in -80 degrees centigrade for 24 hours and later transfer it to liquid nitrogen.
Please suggest any care we need to take or modified procedure we need to adopt for the freezing and reviving this particular cell line.
Hi Tej,
The way you are doing same as we do. The only difference is we use 100%FBS for freezing the cells instead of media and its perfectly fine.
Ram
I am using regularly to freeze and thawing saos-2 cells in my lab.
The procedure of mine is:
50% media + 50% FBS+5% DMSO-----filter and preserve at -200C.
Cells were trypsinized, centrifuged and decand the supernatant and add 5 ml crymedia from one T-25 culture flask cells. Aliqot in 5 tubes.
Storage procedure:
1st put in freezer at 00C for 30 min, then at -20 0C for 1 hr, overnight at -800C and finally in liquid nitrogen.
For thawing,
add 1 ml media in cryo-cells and then centrifuge at 1000rpm for 2 min, decand the supernatant and tranfer the cells in flask with media and put into incubator .
Good luck.
Follow the ATCC protocol attached by Enrico. These cells are pretty hearty. It takes a lot to damage/kill them.
Hi, we usually add 20% FBS and 10% DMSO at a final concentration for freezing. We let the cell at -80°C overnight before transferring to liquid nitrogen. When you thaw the cells, be sure to make it as fast as possible and feed them with 20% FBS. After 3 hours, your cells should have attached. You can than change the media to your regular media with 5 to 10% FBS.
Hi, we use 90%FBS+10%DMSO for freezing. Keep in isopropanol box at -80 C for 1 day, then transfer it to liquid nitrogen.
For thawing, we directly keep the vial at 37 C water bath.