Dear Gemma Nomdedeu-Sancho

it is tricky to rescue stressed out iPSCs but worth giving a shot if this was the only frozen vial you had.

After thawing , let the cells recover but in this time you need to scratch out as many differentiated regions as possible. Mark the differentiated regions under the microscope and scratch them away with pipette tips, suck out the medium and add fresh medium. Repeat this everyday till you can split the iPSCs.

After the first split if the cells begin to grow normally then you are on the lucky side. Otherwise, the only other option would be to mark a good iPSC region and then GENTLY scratch (simultaneously collect ) it in a pipette and seed it onto a next matrix coated plate. The idea would be to pick up "only" the good iPSC colonies and help them to recover and grow in a new cell culture plate.

This youtube video might be helpful- https://www.youtube.com/watch?v=bFjC0InTNm0

(If you do not have access to a microscope under the sterile bench, then a simple solution is to mark the bottom of the well with a lab marker so that you know where you need to scratch when changing medium inside the cell culture bench. This however takes a bit of practice and patience)

Once the iPSCs start to grow well then you can expand them further and of course freeze down more backup aliquots as well.

Good luck !

Thank you so much @Shambhabi Chatterjee for your help!!

With some help from other labs I did both things: scratch the differentiated areas and pick the good IPSC regions. They seem to be growing better passage after passage.

Thank you again for your kind response and your detailed help.

i agree with dissecting undifferentiated pieces or removing differentiated areas. Quick dissection of the undifferentiated pieces would be best for the plates you showed because they were overgrown and cells needed fresh dishes to grow well. There is a way to save hPSCs even if the majority of each colony is differentiated. Send me a message, i will send you a protocol. Including how to passage manually and how to cryopreserve. For now - your culture is saved and thats good.