I suggest please try with matrigel also. Also do check the Co2 levels in the incubators using the CO2 sensor, as it happened with us the display of CO2 5 % was not matching with inside levels due to problem in sensor.  

Make sure to add some ROCK inhibitor on the first day of passages or thaws, and give  the geltrex plenty of time to coat before seeding the cells!  As mentioned, matrigel also works, as well as vitronectin. Also make sure you're seeding them not too sparsely-- stem cells like having neighbors.  Good luck! 

I've definitely experienced this in our lab. Density as others have said is the most critical, though I suspect you've tried increasing/optimizing this already. Sometimes with tricky cell lines I will thaw on MEFs and transition them to feeder free (preferably matrigel for me but geltrex is probably equivalent). One or two passages on MEFs is all it usually takes.

Laminin 521 works well for iPS cells attachment and it has the advantage over Matrigel and MEFs of being defined and xeno free (if you need to).  Hope this helps

https://www.stemcell.com/celladhere-laminin-521.html

ROCK inhibitor is extremely beneficial for promoting the attachment and survival of freshly thawed pluripotent cells.  Y-27632 is a highly potent and selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK).  Used at a concentration of 10uM with overnight incubation when thawing or passaging ESC or iPSC, Y-27632 enhances the attachment and survival of dissociated ES and iPS cells by preventing dissociation-induced apoptosis.

coat plates with geltrex for longer period of time then recommended(O/N). Wash cells twice when passaging. Recommend to use EDTA method for passaging and works best for me. Pipet out geltrex just before plating cells

You can try growing your iPSCs on iMEFs with the classical hESCs medium (20% KO Serum Replacement based). It is definitely right to add ROCKi too. But the incubator health is the first thing to check. Calibrate your incubators for CO2, temperature and humidity. If it is a water-jacket based system, check the water amount there. The other tips are: decreasing the times of keeping mediums out of refrigerator, keeping them protected from light and worming them up at RT for a shortest time. Regarding the thaw: Try to thaw cells directly to iMEFs in KO SR medium. On the next day at the medium change, do not throw the floating non attached cells/clumps. Gently transfer them to the new iMEFs containing well and add 25% of fresh medium. Keep both wells for at least one week with a low medium volume (1.5ml per well of 6 well plate). Good Luck and let us know the progress.

Thank you very much all of your recommendations!! 

I have been trying most of your suggestions but iMEF. I am not very sure if we have got iMEF in our lab. But I will try! I haven't measure % of CO2 manually, that's a very nice point, although I have tried 2 different incubators in the same room and 1 incubator in another cell culture room, they never ever attached and died! And I have tested them for any possible contamination and all the results are negative.

The problem is we haven't changed anything in our culture room, Rocki has always been used after passaging/thawing, geltex coating was as usual, we only use EDTA since they are in colony, medium has always been in dark and RT when it will be used (like an hour)...

They have always been growing very nicely, we have never had any problems until now. I am now scared to thaw a new vial!

We had a similar problem for some time. Suddenly, one day, cells started to detach after thawing or passaging. In the end, it was a problem of the coating. We are (and were) using Matrigel. It was just a problem of handling the Matrigel when coating the plates. Make sure that you coat properly the plates and that you don't wait too much before using them.

Good luck!

Hi there,

Why don't you try Matrigel? The cells should attach very well to this material.

While passaging the cells, it is also necessary that using EDTA, your cells shouldn't detach completely from the substrate but tend to detach. Incubating more than this would cause the problem you have.

Are you freezing the iPSCs in the Bambanker freezing media?

Good luck

Using Rock inhibitor at the day of seeding (not afterwards) is essential. Also you have to give enough time for the coating. (usually for Matrigel it is 30-60 minutes minimum and stable for 2 weeks).

Hi,

Cell culture is badly managed. Matrigel is dedicated to ESC Medium. Vitronectin to E8 Medium. Culture iPS cells on rh vitronectin. Passage iPS cells with 0.5 mM EDTA in colony. Wash  iPS cells with dPBS before EDTA will be added.

Good luck!

Hm. I'm having the same set of problems. We found part of of our problem was residual TrypLE in our culture, but the problem has reared its head again. Was this figured out for you?

We were facing the same problem sometime back. Check if your plastic wares have been changed to different company and if they are "Tissue Culture Treated" cause this plays a very important role. We had bought some plastic ware which were chemically treated which caused the iPSC not to attach/detach.

The second possibility can be to check if your other cells are growing fine and to monitor the CO2 levels in your incubator.

Several people have suggested the use of matrigel, apparently not realizing that Geltrex is matrigel.

For those recommending vitronectin, there is very little difference in performance between vitronectin and Geltrex for the culture of iPSCs.

I wonder if the problem might be insufficient coating of Geltrex? I have seen here that many people apparently don't check the concentration of their matrigel and simply use a standard dilution ratio. Ideally you should use a standard mass of protein per coated area. I routinely use 1mg per 100mm plate equivalent.

Also, Geltrex/matrigel must be prepared properly. Unless you prepped it yourself, you cannot trust that it was done correctly. I would suggest that you try a new lot, or at least a new prep, of your Geltrex.

Hi,

1)Add rock inhibitor to matrigel containing media(O/N). 2) Wash iPS cells in medium containing rock inhibitor.if possible wash cells 2x 3)Resuspend cells in medium containing ROCK inhibitor and keep volume of only 1.5ml/ each well of six well plate O/N and add additional medium the next day. This definitely should help attach cells to plate surface in 2-3 hrs and will see spreading as well, otherwise chk. viability of your cells.

Harnath Shelat, she already is using ROCK inhibitor.

In my opinion, for H9 cells, the matrigel coating quality is very critical as much as cell density. Additionally, room temperature is critical since cellular stress increases with dramatic temperature differences.

Hi,

I am facing a problem with the iPSC cell culture line, to the cultures ROCK inhibitors are added and they are grown on matrigel, in mTeSR1 media. after thawing or after passaging each time there is 50% cell death and this happens every time and cells get deattached very often. kindly help me to troubleshoot the problem.

Dear Tuğçe Munise Şatır did you have any improvement with your iPS cell culture? I'm struggling with a similar issue with a specific cell line. After passaging with dispase the cells didn't attach anymore. I already tried adding more matrigel and more iROCK, 1 well from 12 and 24 well plates, and nothing is working. I think that in my case Dispase is not ideal and is causing some lack of adhesion capability in these lineages.

Best use EDTA treatment and wash 1x with pbs and dislodge cells with small scrapper. This way small clumps of cell will be obtained that is best for splitting and freezing. Will give greater viability when freeze thawed. Good Luck

Dear Harnath Shelat, thank you very much for your advice. In this case, Versene could be an option, am I correct? What is the concentration of EDTA do you mean, 0,5mM in PBS? What is the time of incubation?

0.5mM EDTA. Wash well 2x with dPBS. Add 1.0ml 0.5mM EDTA/1 well of 6well plate. Put plate back in incubator for 1-3 minutes or just leave it under microscope and watch until periphery of iPS colony gets dislodge from plate surface. At that point aspirate EDTA completely and wash well 1x with dPBS. Add 1.0ml of complete medium and either use small scrapper or sterile 1.0ml or 200ul pipette tip to dislodge colonies. Colonies will dislodge easily and can be broken into small colonies with pipetting with media with force.

Treat the plastic with appropriate sticking agent eg poly l lysine,fibronectin,laminate,collagen, or other see cell culture manufacturers or the books Davis animal cell culture:essential methods , or Davis basic cell culture.

An alternative is to adapt/select for cells that are better able to stick down. A few cells may remain attached and then be allowed to grow up. With subsequent passage they may be better able to form a monolayer.