I have thawed 2 vials of cell which were kept at -80C. After the spin cell down, to remove frozen medium, I refilled with a new completed medium and I found some suspicious things which are bigger than my cell.
Could anyone tell me what they are?
PS. I've checked that my completed medium and FBS don't contain these thing.
It is very nice to follow your discussion here, especially you talk about the picture attached. Such a simple photo improves the level of the answers dramatically. In few other questions I'm following, the answers are pretty hypothetical because of not showing of the results. Thus I voted up this question. I strongly suggest to young researchers to show immediately any problem here. In the lab you don't have silly questions, but you often observe researchers are loosing a year of work because of not asking about something was essential.
Looks like some kind of a crystal. Try to dilute your cell suspension with fresh medium and see if these structures become less frequent. It does not look like an aggregation of cells or a contaminating cell type. What kind of freezing medium do you use?
for freezing medium:
10% DMSO
50% FBS
completed medium for the left.
(Completed medium: DMEM with 10%FBS and 1%pen/strep)
Your freezing medium composition is okay. I do not see the necessity for Pen/Strep but it doesn`t harm either. If you just freeze "freezing medium" without cells and thaw it, do you see the same structures?
If the structures (in your thawed cell suspension) disappear after continued culture I would ignore it, if not try gradient centrifugation with ficoll to get rid of them.
unfortunatey that I always prepare fresh freezing medium, so every component''ve already change in lot.
Just a suggestion: next time you prepare freeezing medium, use a little extra and freeze without cells, thaw in parallel with the cell containing vials and see if the structures appear again. But this pure curiosity from my side, as long as cells do what they should, you needn`t care.
I totally agree with Erik. It looks like grown cristals, which may not resolve after thawing. Did you see varying amounts, inverse correlating with viability of the cells? If not, no need to be too concerned, should disappear with changing medium.
Could be some sort of contamination in your water supply (dust, metal grit from piping, etc.) which serves as crystallisation seed. In particular, I experienced phosphates to be especially susceptible to forming crystals with contaminations.
My estimated guess is, it should disappear using a very different batch of FBS.
You should be extremely worried if such a vast amount of fungal spores appears in your cell culture. I am quite sure these structures are no spores, but continuing culture will soon show outgrowth of filaments if these really fungal origin. My guess is with Robert Eibls suggestion above.
I agree that this looks like a kind of fungal contamination maybe either present in the freezing mix or in the culture before freezing itself.
Dear Wareerat,you need to microscopically examine your freeze mix to see if there are these spores prevalent,if not you need to check the vials that you have frozen down.
regards
You could put a few drops of each component of your freezing Medium on a microscopic slide, let it dry and watch the (postulated) crystals develop. Those looking similar may identify the source, for example, if it not the protein containing FBS, but the media, then the crystals are some salt crystals (like I think I remember from PBS drying out), otherwise it could be, perhaps, also some protein agglomeration. Make sure you use 1x of your media (not 10x) in the preparation of your freezing medium.
The large circular pie shaped things look like crystals to me, they must have formed during cell freezing. I have never seen that in thawed cells, I would use fresh media and a new batch of FBS the next time you want to freeze down the cells.
By the way, once you washed the cells with fresh media did the cells grow okay?
Hi. I've never seen this phenonemon before but I agree they are probably protein crystals. What worries me is that you're keeping your cells at -80 which is above the glass transition point. You should store your cells in a liquid nitrogen cooled vessel (below -137 deg C ). Above this temperature you will see reduction in cell viability and physical and chemical changes in the content of your vials over time.
It is very nice to follow your discussion here, especially you talk about the picture attached. Such a simple photo improves the level of the answers dramatically. In few other questions I'm following, the answers are pretty hypothetical because of not showing of the results. Thus I voted up this question. I strongly suggest to young researchers to show immediately any problem here. In the lab you don't have silly questions, but you often observe researchers are loosing a year of work because of not asking about something was essential.
I agree with strongly with Jan`s (Piwowarski) comment above! Show your unexplained phenomena at RG with pictures or diagrams and there will very probably be someone who knows a way to solve it or at least help develop solving strategies. There is hardly any stupid question, but its stupid not to try to ask first.
So, after two weeks, what are these structures, Wareerat? Organism or chemical phenomenon? I would really like to hear about the outcome!!
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