Congratulations for getting your iPSC colonies.

You don't really have to get pure iPSC within one passage, it will be good if you do but not critical. Usually the iPSC chunks on the periphery will have fibroblast attached to it. You just need to keep focusing on the chunks from the center of the colony. Within 2-3 passages you will likely to remove the differentiated cells/carry over fibroblast as long as you pick good iPSC colonies.

I usually stick to mechanical passaging until the iPSC colonies free from fibroblast/differentiated cells, it wont take that long.

Thanks yanuar.

That's helpful.

Cheers!

Hi Ajay,

It is a little tricky!

First of all, only select good looking colonies during mechanical split: sharp borders, dense and round, small cytoplasm/noucleous ratio and etc.

Second, always do low density cell seeding in all passeges.

Third, the coating is critical, if you use Vitronectin, be generous,!

Forth, media refreshing should be ontime and always equilibrate the media before exchange!

Good luck!

Massoud

Dear Massoud,

That is really helpful. I use matrigel and 3T3 feeder layer (separately, not together).

Thanks,

Regards

Try feeder free conditio. It is very useful. You can use truncated Vitronectin for coating. Price is reasonable and the results are nice. I compared matrigel and Vitronectin, the later was better.

good luck,

massoud

Yaa. The kit also describes using vitronectin for feeder free culture. We will order that. So is it possible at any stage to transfer colonies from feeder layer/matrigel to vitronectin or it need some acclimatization ?

Thanks!

It is better to start feeder free but you can switch whenever you want. To get rid of sendi virus you have to do consecutive passages for 6-10 times. Good luck!

Thanks dear. That's really helpful.

Cheers!

It is tricky to obtain the a pure iPSC population on your first passage. Try performing TRA-1-60 live staining and mark the fully reprogrammed stained colonies before passage.

Also try to seed at a low density and perform passage manually during the initial few passages at least to get a pure pluripotent population.

You can also try out Stem cell technology's Relesr reagent which is known to selectively get lift pluripotent cells during passage.