TITLE: Thawing of human pluripotent stem cells    
AUTHORS: Ralitsa Madsen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the thawing of human pluripotent stem cells. </div></div>

[STEPS]
?. If the cells were frozen from 1/2 or 1/3 of a subconfluent (80-90 %) 6-well plate, they will need to be thawed into 1 or max 2 wells of a Geltrex-coated 6-well plate (number of wells might require empirical optimisation based on knowledge about the particular cell line's behaviour). Therefore prepare the required amount of E8/F medium in 50 ml Falcon tubes (always make aliquots so that the stock bottle of medium can be kept in the fridge - max 14 days following supplementation).Supplement the aliquot with RevitaCell Supplement (this needs to be thawed in the dark in advance and aliquotted into smaller aliquots of 500 μl - stored at -20C). As an example how to dilute RevitaCell (100X) in E8/F: add 500 μl of RevitaCell to 50 ml of E8/F; mix well and label as E8/F+R.Make sure to have both E8/F and E8/F+R aliquots ready.Allow the medium to equilibrate to room temperature before use. Do not keep out at room temperature for more than 3 hours without using. It is also a good idea to switch the safety cabinet light off if not in use so that the medium with RevitaCell is not exposed to light. At the same time, take Geltrex-coated plates out to equilibrate at room temperature minimum 30 minutes before use. For coating, see:
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?. [Prepare media reagents and plates for seeding]
Prepare the complete E8/F medium: transfer 10.2 ml of the thawed 50X supplement to 500 ml of E8/F basal medium. Mix well and write date of reconstitution. The medium is stable for 14 days if kept in the fridge. NEVER warm the stock medium in a waterbath.
?. [Thaw the cells]
Prepare 2x Falcon tubes (15 ml) per cell line; these will be used to avoid double-dipping into the working medium aliquot during cryovial washes. Add 5 ml of E8/F (Revita not needed) to each one of the tubes.
?. [Thaw the cells]
Transfer the cryovial  into the cell culture incubator at   and leave for   minutes until a single ice lump remains. Do not use waterbath to avoid contamination! Alternatively, squeeze tight in your hands until a single ice lump remains.
[Incubator, NOT waterbath]
[(approximately)]
?. [Thaw the cells]
Gently mix 1 ml E8/F from one of the 15 ml Falcons to the cryovial with thawed cells, taking care not to spill over. Transfer the suspension to one of the 15 ml Falcons.  Using the same pipette, transfer medium from the 15 Falcon without cells to the cryovial to rinse any remaining cells off and combine with the cell suspension. Mix with the remaining medium in the Falcon without cells. The final volume in the 15 ml Falcon with cells should be 11 ml; discard the empty Falcon tube.
NB: Very important to be gentle and avoid overpipetting. Failure to do this properly may compromise the quality of the cells and result in increased differentiation. All medium should be added very slowly to the thawed cells to avoid osmotic/temperature shock. Try to rock/swirl the Falcon tube in between your fingers while adding medium to the cells in the specified mixing steps.ONLY use stripettes to handle the cells at this stage. They are very fragile and using filter pipette tips will result in high shear stress and potentially break the clumps up too much.
?. [Thaw the cells]
Centrifuge the cells for 3 minutes at  .
?. [Thaw the cells]
Aspirate the supernatant, leaving only 100-200 μl behind. Gently flick the tube to resuspend the pellet.
?. [Thaw the cells]
Gently resuspend the cells in the required volume of E8/F+R for seeding into either one or two 6-wells, calculating for 2 ml per 6-well.
NB: Essential to be gentle, yet resuspend the cells sufficiently to make sure that the clumps are of the right size. This becomes easy to see by eye with some practice. Otherwise, assess clump size under the microscope following seeding and refine if necessary.
?. [Thaw the cells]
Aspirate the residual Geltrex liquid from the coated wells and add the required volume of cell suspension to each well. Check under the microscope that the clumps have the right size (50-200 μm) - the RevitaCell enables cell survival as single cells, so it is more important to get most clumps below 200 μm than to avoid single cells. If required, break the clumps up further using P1000, but be gentle when pipetting (usually 3-4 times of gentle pipetting is enough). If you deem the cells to be too sparse, consider pooling into a single well (when in doubt in such cases, start by resuspending the cell pellet in the previous step in 3 ml medium, then seed 1.5 ml of the suspension into each one of two wells, assess under the microscope and decide whether to pool into a single well in a final volume of 3 ml).
?. [Thaw the cells]
Return the cells to the incubator  and assess 24h later (try to keep a routine schedule where cells are handled at roughly the same time of day). Make sure to distribute the cells evenly across the well by moving the plate up-down-left-right when in the incubator.
37 °C
It is important that the cells are left undisturbed when thawed so they can settle properly.Replenish with fresh E8/F (2 ml per 6-well) medium the next day; NB: NO Revita (but only if the cells/colonies look sufficently large and healthy, otherwise replenish with E8/F+R for another 24h). Note that once Revita is removed, the colonies will exhibit signs of stress on the following day, with several colonies appearing apoptotic and perhaps detaching. This should normalise and the cells should be ready to split again 4-5 days later when 70-90 % confluent (ideally 80 %). The splitting ratio for each line will have to be determined empirically, but usually the first split after thawing has to be gentler than usual.