My experience is, that thawing of IPS is allways difficult, even when you use ROCK inhibitor. Sometimes clones I thaw in the past need up to 6 weeks to became good again.

# suggested this protocol to Prepare freezing media and cryovials 

For Solutions and Reagents Required : 

DMSO (Sigma Ref. D-2650)

KOSR (Invitrogen Ref.10828-028)

For  Method :

1. Mix 0.1ml DMSO and 0.9ml KOSR per vial (multiply by the number

of vials you wish to freeze)

2. Label cryovials with (i) for induced PS cells, name of the cell line from

3.Aspirate media from iPSCs pellet and carefully add 1ml of freezing media per

vial and mix gently once

4. Place the cryovials in the Mr Frosty.

5. Transfer Mr Frosty to -80°C freezer and store overnight

6. After 24 hrs transfer cryovials into the liquid nitrogen for long-term storage

and very important  , work as fast as possible – DMSO is toxic for cells and cells should not be in contact with freezing medium longer than 3 min before transferring them into -80C 

Ref {Human induced Pluripotent Stem Cells Protocols }

Cambridge Biomedical Research Centre

Aleksey,

All these issues are very important (what the Sven and Saleh wrote) and, in my oponion,  another important issue in this case, regardless of the chosen protocol, is the cell density that you will freeze, due to the cell size and their water content.

I suggest you (If you have enough iPSC) test different densities of freezing/thawing.

I only use ROCK inhibitor for preparing near single-cell suspensions for genetic modification experiments or analysis...in other cases, such as freezing and thawing, I don't need it... mechanical or collagenase clumps are much better for freezing/thawing and time to recover the culture is significantly lower....

My freezing media for any pluripotent cell is 90% FBS and 10% DMSO... if clinical grade, then human serum... collagenase clumps are washed in fresh culture medium after decanting them by gravity, and broken a bit by gentle pipetting in the freezing medium before dispensing them in the cryovial and freezing them immediately...

regarding the amount, up to five cryovials have been set up from a well of a six well plate, 70% confluent.. they have recovered the culture in a couple of weeks or earlier...

That is the general rule working for me... obviously the health of the cells before the procedure is crucial... not too overfed and not too underfed... just at the end of the exponential phase and without yellow media...

Thank you for answers!

I'm a bit worried that contact with FBS during freezing would promote hiPSC differentiation.

I'll try several modifications when there'll be enough cells to work with.

Hello there.... I don't have any problems freezing or thawing hiPSCs. 

If it's on feeders, I vitrified but I believe you are asking about feeder free. In this case,  I use Cryostor CS10 (Sigma or Stem Cell Technologies). Normal procedure, detach cells from plate using Dispase (Stem Cell tech), wash cells with normal DMEM - spin down and resuspend in Cryostor, I use Mr.Frosty for 24hr then vials are transferred to LN2.

I use rock inhibitor when I thaw the cells. You will always have some cell death but RY helps to prevent that. By the way, This is a xeno free culture. 

Hope that helps.

cheers.

Hi all,

The best protocol for hiPSC (growing on feeder MEFs) freezing and good thawing afterward is as follows for 6-well plate/ per well:

Such the hES medoim from the wells.

Wash once your cells with DMEM/F12 = 1 ml per well

Add 300 ul (for 6-well plate) of Dispase (it is better than any other!)

Put in the 37C incubator for 7-10 min. Lokk at the microscope. the edges of the colonies should be lift-up.

Such the Dispase (don't worry the cells won't detach).

Wash twice with DMEM/F12.

Add hES medium, 1 ml per well. With 1 ml tip dissociate the colonies but not too much you don't want them to be single-cells but rather clamps to create again colonies.

Transfer to 15 ml tube.

Centriguge 5 min, 1600 rpm in a standart table-top centrifuge

Such teh medium, watch the pellet.

Resuspend in 1 ml freezing medium (hES+10% DMSO) and transfer to cryovials.

I don't use at all ROCKi (only when I perform protocols with single cells after accutase), Some cell lines become "addicted" to ROCKi and they can not grow well without it.

Good Luck,

Dorit

We also don't have major problems with freezing and thawing human iPSCs. We have best results with culturing cells in feeder-free conditions (on vitronectin- or matrigel-coated plates; mostly in a 6-well format) in E8 or mTeSR medium until cells reach about 70% confluency. These cells are than detached using EDTA (or Versene) into small clusters, which are then resuspended in E8 medium containing 10%DMSO and frozen in cryovials (usually cells from one well of a 6-well plate in one cryovial) first in MrFrosty overnight and than transferred to N2. We do NOT use ROCK inhibitor or any other small molecules for freezing or thawing.

What Tomo wrote is right in that point that we don't use ROCK inhibitor at the time I worked in Tomo's lab. I just mentioned this because of Aleksei wrote and I know that some people tried this but as Aleksei also - without better results.

Hi all,

What would you suggest for iPSCs grown on a MEF feeder layer?

ROCK in the thawing medium which can be just serum, but not in the culture after thawing, definitely.

for MEF layers, recommbinant trypsin should separate human ips colonies from feeders... a trick that always worked for me was TrypLE without washing just after media aspiration and small volume just 1/10 of the culture volume.... three to five minutes RT meanwhile tubes are prepared with one volume of complete culture media each three wells in a 15 ml conic...  the media from the tube is aspirated and dispensed over the TrypLE and with gentle pipetting up and down to the tube again...

now, gravity!  let the clumps of pluripotent stem cells decant, and aspirate the supernatant with the feeders...  with new media, more pipetting will produce near-single cell suspensions

the most efficient way of separating colonies from mefs is collagenase, though... detaches colonies and leaves feders on the plates...

Thanks for your answer Maria. What concentration of ROCK inhibitor do you think is best?