8 replies to this topic

[mrscientist]

human mesenchymal stem cells purchased at passage 1. initially expanded fine, then successfully passaged with >4 population doublings before freezing. upon thawing, cells look okay for a few days, then round up/detach/die around days 4-6 with no distinct evidence of contamination. occurs from two independent donor sources. cells were cultured (media/16.5% fbs, though now attempting with 10% fbs and filtering media), passaged, frozen (30% fbs/5% dmso) and thawed (37 water bath and centrifuged, though now attempting without centrifuge) per supplier's recommendations. same media/fbs used for another cell line with fine results.

i'm not looking for a discussion/debate on freezing/thawing. i'm simply wondering if something did happen during freezing/thawing, is it possible for the cells look ok for awhile before dying, or is something else happening? tia

Edited by mrscientist, 27 February 2012 - 12:09 PM.

[bob1]

I haven't worked with "stem cells" before other than primary MEFS and skin cultures, but it could be that the cells are getting a little confluent post-thawing which may halt growth.  It could also be that there is a need for some extra additive in the culture that is missing, though from your post that you have at least followed the manufacturer's instructions, so this is a little unlikely.

Some cell lines are particularly sensitive to changes in FBS stocks - if you have recently had a new supply come in, then it could be that.

You could try conditioned medium - perhaps from the line that is growing well.  Take a fresh culture of about 50% and add some new medium, leave for 12-24 hours, then remove, filter sterilize and use on your finicky cells.  You can use this diluted to normal medium too.  Store frozen in aliquots at -80 long term, or short term (a few days) in the fridge.

[mrscientist]

hi bob1, thanks for your reply. the cells were quite confluent before freezing, but i'm plating around 1500-2500 cells/cm2 after thawing, which looks like about 30-50% confluency during their 3 day "healthy" phase. they should be able to get back in the growth phase with more room after thawing, right? media i'm using post-thawing is the same as pre-freezing. fbs from the same bottle and media from the same lot.

i'll keep the conditioned medium in mind. though i haven't had problems with this cell type (albeit from a different source) before, so i'd really like to keep things as close to "normal" as possible.

[ravibiosciences]

Just a thought from my side-   I wish your cells were perfectly frozen (working on ice and then to -80C /Over night to Liq N2) and thawn (90sec in water bath), hope you are sure that you used filtered, sterile FBS (have a look at the FBS batch). I have once experienced a problem with endothelial cells growing very badly in dishes of different company. Just try changing the conditions incl other cells as positive control. Good luck.

[mrscientist]

hi ravibiosciences, thanks for your reply. cells were frozen as you stated. just checked fbs lot again and it is sterile, but it was giving good results before freezing anyway.

i did forget to mention i have been using different dishes with the same results.

[bob1]

mrscientist, on 27 February 2012 - 01:05 PM, said:

hi bob1, thanks for your reply. the cells were quite confluent before freezing, but i'm plating around 1500-2500 cells/cm2 after thawing, which looks like about 30-50% confluency during their 3 day "healthy" phase. they should be able to get back in the growth phase with more room after thawing, right?

Unfortunately, many primary cell types, and I suspect, stem cells, are very susceptible to confluency as they are contact inhibited.  In some cell lines this is a reversible process, but in others it causes senescence, which is irreversable.  I don't know about your particular line though.

[mrscientist]

hi bob1, thanks again for your reply. i've "rescued" this type of cell from confluence based senescence before, so i don't think that's (hope that's not) the problem.

i was looking back at some images of the cells and there were quite a bit of floaters upon thawing. in my present attempt, i've thoroughly washed away the floaters to see if maybe the dead ones were killing the live ones (?).

[mrscientist]

the problem was the non-filtered 16.5% fbs in the media. i had four mediums, 10% fbs filtered, 10% fbs unfiltered, 16.5% fbs filtered and 16.5% fbs unfiltered. all of the cells survived and thrived except for the cells in the 16.5% fbs unfiltered media (all cells split from the same flask and equal in number). now my question is why did this happen? the cells in the 10% unfiltered fbs were fine, it was only the cells in the 16.5%  unfiltered fbs that died. what is being filtered out of the high concentration fbs that was killing the cells? not important for my experiments now, since i'll just be using 10% unfiltered fbs, but i'd just like to know.

[bob1]

What sort of filter were you using?  - you can look up the binding characteristics on the Whatman website if you were using a whatman filter.