Hi All,
I would like to check whether commercially available cryovials with silicone gasket, internal thread are safe enough to store in liquid phase of nitrogen tank or not. I see all labs are practicing their own way. They said no problem and can dip into it. Some said no, the liquid can go into no matter with silicone seal or inner or outer thread and suggested to store in vapour stage instead. (above the liquid). Available cryoflex is not also convenient to use as it may take time to do it and still exist tiny spaces for liquid to come in too. Very confusing. What is the best method to seal the vial before putting into the cryobox of LNT. Are there any special seal? How about wax? Aluminium tape? or Silicone paste to wrap. Paper tape?
I disagree with the other answers. If you want to ensure the lowest chance for cross-contamination from one sample into another, you should store the vials in the vapor phase only.
If you must store in liquid phase, which I do not recommend, you can seal the tube using Nunc cryoflex tubing sealed around each cryotube. It adds cost, inconvenience, and the tubing seals with heat which can interfere with optimal cryopreservation.
It has been documented that viruses have transferred between blood samples stored in liquid nitrogen.
And, there is a reason *all* the handbooks, including the handbook from Thermo Fisher who makes the Nunc Cryovials and Nalgene Cryovials, recommend vapor phase storage. Furthermore, the guidelines for human cell therapy applications require vapor phase.
I would argue that one would have no way to know they had a cross-contamination event if they did not test for it. Case-in-point: the extensive contamination of various cell lines with HeLa cells went unrecognized for many years. (Not that it happened via liquid nitrogen. It probably happened in the hoods.) My point is just that low level cross-contamination is probably way more common than scientists want to believe.
BTW, if the tube is not tightened all the way after completely freezing (before putting into liquid nitrogen) the liquid nitrogen can escape upon thawing and the tube may not explode.
I practice other techniques to avoid cross contamination of cell lines with unwanted cell types, bacteria, viruses and mycoplasma such as keeping media bottles specific to each cell line, cleaning the hood and traps completely between cell lines, and only working with one cell line at a time, except for experiments involving multiple cell lines.
Just my input, but I do run a very tight ship.
Good luck!
Jen
I have stored cells in internal thread silicone sealed tubes for 30 years and have had very few problems. one bad batch of tubes where the liquid entered the tube and the seal did not expand to release the gas so a few tubes exploded on thawing but only 2 tubes in about 3000 stored cell lines so far and I think that the problem is less likely to happen if all of the aqueous sample is at the bottom of the tube and not covering part of the seal which can happen if the cells are frozen not in the vertical position
Hello! We use vials similar to what you describe on top.... from Nalgene. We slow freeze them on -80 and then take to the NT. Haven't had a problem.
Best
Yes it should be fine, have done it many times. Just buy good quality vials such as nalgene.
Thank you Paul Rutland, B. C. Menarim and Paul Griffin. So I can conclude that Web info of commercial companies recommendations are not really necessary. They made me confused.
So, it is good enough to use proper cryovials and just dip into liquid phase of LNT. Not necessary to use Cryoflex and keep vapor phase in LNT. Thank you.
We have been using Nalgene cryovials since a long time and haven't faced any problems. We store in liquid nitrogen.
Thank you. I am safe. All info on web, made me worried if I did wrong way. All other people in nearby lab also do the same things. Just curious to know how people in other parts of the world are doing. It seems like no one concern even about vapor phase and liquid phase in LNT. We can just simply dip it in. Correct me if I am wrong. Thanks.
I disagree with the other answers. If you want to ensure the lowest chance for cross-contamination from one sample into another, you should store the vials in the vapor phase only.
If you must store in liquid phase, which I do not recommend, you can seal the tube using Nunc cryoflex tubing sealed around each cryotube. It adds cost, inconvenience, and the tubing seals with heat which can interfere with optimal cryopreservation.
It has been documented that viruses have transferred between blood samples stored in liquid nitrogen.
And, there is a reason *all* the handbooks, including the handbook from Thermo Fisher who makes the Nunc Cryovials and Nalgene Cryovials, recommend vapor phase storage. Furthermore, the guidelines for human cell therapy applications require vapor phase.
I would argue that one would have no way to know they had a cross-contamination event if they did not test for it. Case-in-point: the extensive contamination of various cell lines with HeLa cells went unrecognized for many years. (Not that it happened via liquid nitrogen. It probably happened in the hoods.) My point is just that low level cross-contamination is probably way more common than scientists want to believe.
BTW, if the tube is not tightened all the way after completely freezing (before putting into liquid nitrogen) the liquid nitrogen can escape upon thawing and the tube may not explode.
I practice other techniques to avoid cross contamination of cell lines with unwanted cell types, bacteria, viruses and mycoplasma such as keeping media bottles specific to each cell line, cleaning the hood and traps completely between cell lines, and only working with one cell line at a time, except for experiments involving multiple cell lines.
Just my input, but I do run a very tight ship.
Good luck!
Jen
We cryopreserved the samples using cryo vials and there after sealing it with
Parafilm Moisture Proof Sealing Film (laboratory sealing film paraffin).
Anjali Somal> Parafilm does not work at all. Once you dip it into LNT, your parafilm will be broken. Do not do it. It is just waste of money and time. I noted other labs are doing the same and they never check back what happened to parafilm after they dip the sealed tube into LNT.
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