Thanks for your information. I know there are a few different matrigels, this one is supposedly for hiPS cells, though it arrrived before i started at the lab. The medium is the same medium the lab that sent us the cells used, so i'd rather not change it. All i do now is just give them media daily :) any idea on ways to remove differentiation? Now i just try to mark an area using a microscope and gently scrape to remove as much as possible, but this only works for larger areas and not close to a colony. 

Hi Leontien,

For picking you can make a "grid" over the colony with a pipette tip (P10) and then lift the small pieces, in this way you can also take only the center of the colony and get rid of the differentiated parts (you might need a microscope in the flow hood). You may also add ROCK inhibitor (Y27) to your culture when you passage it, we use 1:1000 but  you may increase it a bit if you cultures look worse than usual.

Good luck,

Renata

Thanks! We don't have the option of a microscope in the flow hood, would be cool to have an EVOS in the hood :) I'll try the 'grid' version next time. I used the ROCK inhibitor only after thawing. 

When you plate the cells in low densities use ROCK inhibitor to prevent cell differentiation and/or death. With higher densities you should be fine without any. Cell passaging can be done using TrypLE, Accutase or dPBS + EDTA, I would let them grow till around 70%, more and they start to differentiate.

You could check some E8 papers on details about passaging + medium.

Hope this helps you :)

Thanks! I've been reading the E8 papers and I want to test the passaging using EDTA as well. I'm making a cell bank but have some dishes I can use to optimize the protocol. Has anyone by any chance compared E8 and mTeSR1 medium? 

I have added some links to handy E8 papers and one to a similar question that you have! Hope it all works out!

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084903/

http://www.ncbi.nlm.nih.gov/pubmed/23099485

https://www.researchgate.net/post/Have_you_seen_a_difference_between_E8_Vitronectin_and_mTESR_Matrigel_for_iPSC_in_terms_of_differentiation_ability_or_chromosomal_stability

Dear Leontien I perfectly understand your situation...was the same for me many years ago! However if I can add something to what the other guys suggested you I would try ReLeSR (from stemcell) to split the cells. This is a dissociation agent that selectively detach only undifferentiated part of the colonies and in a couple of passage allows to get perfect iPSC cultures. I was really sceptic at the beginning, but then I tried it and it worked perfectly without the need to clean the colonies manually (that is very time consuming and not that easy at the beginning). I always add ROCH inhibitor when I split the cells to prevent cell death and differentiation, even if ReLeSR give rise to small clumps and not single cells.

Good luck

Cecilia

I do share your feelings about being the only one in the lab in one field, and not having even a single person to show whether your cells look ok! But you will manage either way, maybe with more time and patience :)

So based on my experience, I would say the colony in the 1st pic looks quite nice, and there is some differentiated cells around, which you might want to clean before splitting (I use ReLeSR from Stem Cell Tech, no need to cut and scrape then). 

2nd pic seems to be more prone to differentiation (I think the elongated cells are already differentiated, most likely neurons). 

So for feeder-free human iPSC culture, I would recommend coating either with matrigel or vitronectin (to which I switched from matrigel, because I find it easier to work with and one does not have to be as rigorous as with matrigel). For splitting, as I mentioned, you could try with ReLeSR, it is an enzyme-free dissociation reagent. Otherwise you could still use a scraper, but in that case rather use 5ml pipette than p1000. And do use ROCK inhibitor on the day you split, it helps attachment and survival of single cells. Next day on, you could continue with regular mTeSR1 (previously I was using mTeSR8 as it has a defined composition as opposed to mTeSR1, yet after several passages cells did not look happy and most of them did not even attach and survive after splitting, hence I switched to mTeSR1). 

You could check the links for vitronectin and ReLeSR if you like to give them a try.

Good luck!

https://www.lifetechnologies.com/order/catalog/product/A14700

http://www.stemcell.com/en/Products/All-Products/ReLeSR.aspx

Thanks guys! I'm looking at the ReLeSR reagent as well, I've tried the EDTA releasing method on an old dish today and see what turns up. I guess it will take some time to get a perfect protocol for our lab working, but I'm getting a better feeling for the cells. The ReLeSR reagent looks really cool :) So it really works like Stemcell promotes it? 

:) It does! It is quite fascinating to see clusters breaking apart from the colonies just in a good size. Try and see it yourself!

Hi Leontien,

the first picture looks good with some differentiation around. The second is not good at all and I would remove it completely. If you have your iPS already in a well of a 6well plate or in dishes you don't need to pick them manually. The manual pickig is usually done when you do the reprogramming starting from fibroblasts since you want to be sure you have single clones. It would be better if you can get a microscope to clean the differentiated colonies since with the scraping without seeing what you're doing you might anytime leave some differentiated cells that will be expanded anytime you are splitting.

When I have to split the cells I use dispase that might be also cheaper than the other reagent you mentioned and it works fine. I was working in a lab where we were making our own iPS medium that can be changed every other day since we had a lot of iPS lines. I am now in a new lab and I use mTesR1. The differentiation sometimes depends on how the clone was initially reprogrammed and picked other times depends on the way you passage the cells. I never use the Rock inhibitor except when i have to start a differentiation ad accutase the cells since they will be in single cells and the iPS don't like to be like that.

Hi Leontien,

All the tips on culturing iPSc in our book: Atlas of Human Pluripotent Stem Cells in Culture. http://www.springer.com/gb/book/9781489975065

We do culture iPSc in mTeSR1 on matrigel. Density is very crucial. We are not far away from Imperial, you welcome any time. Your first image: undifferentiated cells on the left are very good. Second image: they are neural progenitors

Good luck

Hi Leontien,

one more comment: I recently realised iPSC on vitronectin grows slightly slower than on matrigel - although they still remain undifferentiated with normal karyotype, fyi :)

A neat trick to clean up your culture without the need to mechanically removing the differentiated cells (second image) is to us Stem Cell Technologies' ReLeSR: 

http://www.stemcell.com/en/Products/All-Products/ReLeSR.aspx

Very easy to use and you will find that your colonies will improve with each passage and differentiated cells will remain attached to the old plate.

Thank Sun Yung, I have tried ReLeSR but in my hands it didn't give good results. I'd like to thank everyone for the tips and sharing their knowledge with me, these iPScs are growing quite well now! On to the next steps :)

"When you plate the cells in low densities use ROCK inhibitor to prevent cell differentiation and/or death. With higher densities you should be fine without any. Cell passaging can be done using TrypLE, Accutase or dPBS + EDTA, I would let them grow till around 70%, more and they start to differentiate."

Sorry, but this answer makes no sense. Density is not the problem that is resolved by ROCK inhibitor. Clump size is the determinant. You can have a very dense concentration of single cells and they all will die, while larger clumps will survive.

The same is true for susceptibility to differentiation. Percentage of confluency is not the problem. The size and age of the individual colonies is what determines the likelihood of differentiation.