My supervisor has asked me to find a protocol for freezing and thawing naive T cells, with details regarding the % recovery of the T cells once thawed, but I'm struggling to find any information online.
Has anyone got an experience with freezing and thawing naive T cells?
Hi
First you should have prepared freeze mix ( 80% AB serum + 20% DMSO) which should be at 4C
Prepare the t cells you need to freeze in a tube with culture medium and the concentration should be 2-20 million / ml
Prepare cryo vials and their number according to the number of cells you need to freeze but I prefer 1-10 million / cryovial . Label them using permanent marker pen since you can't label them when they are cold
Prepare Mr frosty . (each Mr frosty can take 18 cryo vial) keep Mr frosty at room temperature
Prepare a basket filled with ice and put the cryovials , the cells tube and freeze mix inside it for not less than 15 min to make them cold enough
Work inside hood
Open one cryovial , add 0.5 ml cells using pipette then add equal volume of freeze mix using pipette (if you add 0.6 ml cells add 0.6 ml freeze mix) slowly drop by drop with little shaking for the vial . Beware not to spill it and then close the lid . Put the vial inside ice
Do the same for the rest cryovials and when you finish 18 cryovials put them inside the Mr Frosty . Close it and then
Put it in -80 freezer for 3 hours and then transfer to -150 freezer or put in liquid nitrogen ( for long storage)
In Thawing
First prepare big tube like 20 -50 ml filled with culture medium containing not less than 10 ml medium , pasture pipettes , a big tube filled with culture medium ,water bath at 28C
Take Mr frosty out of the freezer open it and put the cryo vial in the water bath in a special rack so that the lid would not be covered with water. Wait till there is thawing in the upper quarter (don’t open the lid , just look at bubbles which may form ) and then take it inside hood , open the lid and using pasture pipette add few drops of medium to the vial and wait till good thawing occur in all the vial then take all the vial content using the pasture pipette and pour it into the 10 ml tube culture medium ,close the tube and then spin at 1500 rpm 5 min brake 1
Take the tube out of the centrifuge, take care not to shake and so lose the palette. Pour the medium of the tube and then close it and flick the bottom (to move the pellet) and then ad 10 ml of new medium and shake well and then do the counting
Using this protocol you may be able to retrieve 50-60% of the frozen cells sometimes you may be lucky to retrieve 80%
Regards
To add to what Zaid said, I typically use benzonase in my thawing buffer to decrease the stickiness caused from DNA being spilled by lysing cells. I do my spins at 200g for 10 minutes, room temp, brake
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