Basic Lab Procedures: Freezing and Thawing cells into/from cryowells

Oftentimes in lab, situation arises when you need to store cells for future use. It is possible to store the cells without freezing them, but has many disadvantages like having to change the media regularly and the increase in passage of the cell or the like. So since the technology became available, freezing and thawing cells have been part of a very routine procedures in bio-tech labs.

Now what do we need for freezing and thawing the cells?

For thawing:
not much really... XD just your body heat will suffice.
but the chances are that you would want to plate the cells on to a T25 for something, so for that you would need
-a plate (t25)
-some media
-some tubes like 15ml falcon
and basic lab machineries like pipets, hood, and centrifuge.

For Freezing:
Need a little bit for this one :D
you would need
-trypsin (generally 0.25%)
-media
to harvest the cells from a plate and
-freezing media ( 10% DMSO, 90% FBS )
-cryowell
-cryo tub
and fridge/freezer, and liquid nitrogen freezer.

Now the How To part.

I'll start with freezing the cells.

-First, get the cells you want to freeze from the incubator.
-trysonize it to get the cells off the plate.
-once the cells are afloat, neutralize the trypsin with some media.
-centrifuge the tube(with cell/trypsin/media) at 1800 rpm for 5 min.
-pour out the liquid, while being careful not to disturb the pellet of cells.
-disturb the pellet and add freezing media. (add 1ml for each cryowell you'll be storing the cells, the number of cryowells would depend on how many cells are being frozen)
-mix the solution thoroughly to get a good suspension than add 1ml of the mix to each cryowell. (its specified as 1ml because the cryowells I've used have the capacity of 1ml, there are wells with different capacities, so note this.)
-put the cryowells in the cryo tub filled with isopropyl, and put the tub in -20'C
-after 30min move the tub to -80'C freezer.
-the day after move the crywells to the liquid nitrogen freezer (which is at -196'C)

NOTE: The cells are moved from freezer to freezer because if the cells freeze too fast the crystals that form will be sharp, causing the cells to rupture or lise. DMSO is included in the media to prevent this; however, the cells still have to be frozen slowly. For the same reason, the cryowells are placed in the alcohol tub. isopropyl has a property of freezing very slowly and steadily.

Now to the thawing part.
This is much easier :D
-retrieve the cells from the niquid nitrogen (be careful not to get burned by liquid nitrogen)
-thaw the cells by leaving the crywells in the water bath for a short while (or just hold the well)
-well there is still a small ice formation left, take the crywells out of the water bath and move them from the wells to a 15ml falcon tube (or any other centrifugable tube).
-Add few mls of media to the tube, and spin in at 1800 rpm for 5 min.
-discard the liquid, disturb the pellet and add appropriate amount of media to be used to plate the cells.

DONE :D

I'll be sure to get some pictures for this, I forgot D: