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Thread: cell cryopreservation in -80

  1. #1 cell cryopreservation in -80 
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    hi, there will have any consequences on my peripheral blood mononuclear cells if i transfert them from nitrogen liquid to -80.
    please help me because liquid nitrogen is very expensive for my laboratory
    thanks for helps


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  3. #2 Re: cell cryopreservation in -80 
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    Quote Originally Posted by sambo6
    hi, there will have any consequences on my peripheral blood mononuclear cells if i transfert them from nitrogen liquid to -80.
    please help me because liquid nitrogen is very expensive for my laboratory
    thanks for helps
    You need to provide a bit more info. What sort of media are your PBMC suspended in? Are you using DMSO or perhaps some other cryoprotectant? My general feeling is that the temperature increase is going to negatively impact on your cell recovery rate as well as the useful preservation time, but the extent may depend on a number of factors you haven't mentioned.


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  4. #3  
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    the composition of medium is for 50 ml: 43,5 ml RPMI + 5 ml FBS + O,5 ml MEM + o,5 ml L-GLUTAMINE + O,5 ml PENICILLIN-STREPTOMYCIN.
    After the counting,we divide the number of cells by 20 million for knowing the size of medium and DMSO use to freeze 20 million lymphocytes per cryovial.We supplement the medium with DMSO 20%.; for e.g for 1 ml of medium we supplement 1 ml of DMSO 20%.after,Cells having been put at the -80C during 72H, before to transfert in nitrogen liquid.
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  5. #4  
    Moderator Moderator TheBiologista's Avatar
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    Quote Originally Posted by sambo6
    the composition of medium is for 50 ml: 43,5 ml RPMI + 5 ml FBS + O,5 ml MEM + o,5 ml L-GLUTAMINE + O,5 ml PENICILLIN-STREPTOMYCIN.
    After the counting,we divide the number of cells by 20 million for knowing the size of medium and DMSO use to freeze 20 million lymphocytes per cryovial.We supplement the medium with DMSO 20%.; for e.g for 1 ml of medium we supplement 1 ml of DMSO 20%.after,Cells having been put at the -80C during 72H, before to transfert in nitrogen liquid.
    Okay, aside from the MEM that's pretty much the same medium I use for PBMC. I find I can easily get a >50% viable recovery of cell numbers after a couple of years from nitrogen. But I've never seen a protocol for continuous storage at -80 so I'm not sure what effect you could expect to have on viability. The general procedure for cryopreservation is "freeze slow, thaw fast". The main reason for the fast thaw I've been told is to limit cell contact with liquid DMSO. Whether that counts for a warming to -80oC I couldn't say. I would guess you can expect a negative impact on cell recovery yield and cell viability. You may have trouble resuspending cells during recovery due to cell debris.

    I had a look around for some solid data on PBMC cryostorage but didn't come up with much. My advice would be to do a mini experiment. Start out with some fresh PBMC and try a few different freezing and storage temperatures, including 80-->N2-->80. Look for cell recovery/viability by EB/AO staining and maybe try some FACS for forward/side scatter. If you have any FACS stains for apoptosis maybe try those too.
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