I need to transport cells from one country to another for further studies but is essential to preserve the viability.
You need to freeze cells and ship it in dry ice. To freeze cells, mix 5% or 10% of DMSO in 90-95% human serum (for human cells) and do slow freezing in a "mister freeze" or in a box fill with hydrophobic cotton. Autologous serum is the better. Decomplement it by heating 20min at 56°C before using with cells. 1M to 5M per mL and tube is a good concentration. Above might reduce viability.
Hi Javier,
I agree you should freeze your lymphocytes and ship them in dry ice. The method of freezing cells seems to vary from one lab to another but mine uses a freeze mix of 12% DMSO with 40% serum (whichever type you are going to culture them in later) made up in 48% culture media (again whichever you are going to culture them in later).
The trick is to make the freeze mix very carefully on ice. Add DMSO dropwise to half of your serum already cooled on ice (it's an exothermic reaction so will denature the protein if you do it too quickly). Do not do it the otherway round as the DMSO will freeze. Then pipette in your media, slowly at first then more quickly. Then add the rest of your serum to the cooled freeze mix.
Prepare your lymphocytes at a concentration of 5 to 10 million cells per mL (ideally) in culture media with 20% serum. Put this on ice until cool, then dropwise add an equal volume of freeze mix, stopping to shake-mix periodically. Every 10 sec or so you can increase the rate of your drops. Continue until the whole lot is mixed. Alequot as rapidly as possible into pre-prepared cryovials and put in a 'Mr Frosty' (or large polystyrene box filled with tissue paper) and place in the -80oC to freeze slowly over 2 days. Then transfer to liquid nitrogen until you are ready to ship.
It depends on how long the cells will be in shipping. I've transported live cells in a culture flask from South Africa to the UK, but it is best if they arrive within 48 hours. You can grow the cells in culture flasks (T25 or T75), and before shipment fill up the flask with culture medium and seal with parafilm. Package in a box or container to prevent too much movement. When they arrrive, they can be centrifuged and cultured as previously.
hello all, I'm a beginner in these assays so I tried things. I have to do assays in a country and then, in one year later, will send the frozen PBMC to another coutry.
I first tried to freeze in SVF +20%DMSO. The viability is correct but, it is difficult to resuspend the pellet after thawing. So I've tried RPMI+20% SVF+10%DMSO. After thawing, we remarquably recover more cells and the viability is equal.
after 2 days at -80°C in a mister freeze I transfert in liquide nitrogen,
So, that will be our protocol of freezing. For the transportation, I do not trust the dry ice packaging so we'll use a nitrogen transport tank. there is a foam inside. we put liquide nitrogen for 24h and then thraw it (liquide nitrogen is forbidden in planes transportation). The vapor inside let the temperature at the same temperature of the liquide one for 3 days of transport.
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