What do the colonies look like? Would you want to share some pictures on here so that we can take a look? And what's the current passage number and the passaging method you're using? It's a good thing that you're keeping the split ratio at 1:1, maybe you can try shortening the split interval slightly, as long as you can maintain an optimal size of the clumps for passaging. You can also try recombinant vitronectin alongside Matrigel, they may stick to it a bit harder, that is if you think you can split them into 2 wells at some point (or use smaller wells like those of a 12-well plate).

But I'd be worried that whatever happened to this cell line may have compromised its pluripotency and it would probably be difficult for you to dedicate like a 100'000 cells for flow analysis of TRA antigen expression.

Thank you very much for your response! This is what my colonies typically looked like the day before they lifted. They're at passage 25 now and we received them at passage 23.

With the vitronectin do you plate that underneath the matrigel and use the same coating protocols as usual ie. I usually incubate vitronectin at RT for 1hr and matrigel at RT for 1hr?

I might attempt the 12 well idea too.

Thank you again! I'll give these ideas a try. :)

That colony looks beautiful, a perfectly normal PSC morphology. That's so strange they grow so slow. Coating underneath Matrigel with vitronectin probably wouldn't help so I'd just seed the cells directly on vitronectin, either rhVTN-N from Thermo or VTN-XF from StemCell Tech. Incubating it at RT for an hour is good.

What's your concentration of Matrigel you're using for coating? You passage them as clumps and not as single cells, is that correct?

I have worked with an ESC line that was derived on VTN and I could never make it to grow on Matrigel, the clumps would never attach and the cells would just die, unlike all other PSC lines I used that could seamlessly transition between the two matrices.

I wouldn't want to accuse Miltenyi of not doing a good job with the StemMACS medium but its formulation is not published and I don't think it's as widely used as other media formulations like mTESR-1 (richer) or E8 (low-protein and xeno-free). Next time you're ready to passage them, I think I'd go for the 12-well plate and split the cells into 2 wells, one with your Matrigel and StemMACS medium and the other one with VTN and E8. The cells should be able to make the transition into the new condition relatively seamlessly, with no need for adaptation.

Thank you, it was indeed a beautiful colony which made it all the more heartbreaking when it lifted!

I do passage them as clumps as much as is possible but I'm unsure about the concentration of my matrigel. The information with the matrigel just said to use 11.64ul of matrigel per 1ml of media.

Thank you again for all your responses, I will definitely attempt those two conditions in the 12w plate next time I split. I do have some mTESR 1 in the lab as well so maybe I will attempt this media too. Hopefully I'll get my beautiful colonies back!

Hi Marianne,

that was some good advice from Jaroslav.

Were the cells frozen, or live when you received them? Do you know how they were grown before you got them? hESCs always need a little time to adjust to new culture conditions and to change expression of ECM attachment factors. 2 passages is not very long.

If you are really having a hard time with attachment, you can always try using LN-521 or 511. Works very well. I've adapted several lines from Matrigel to LN521 and VTN.

If you are well and truly having a hard time, you can always use Lonza's expensive but excellent L7 system.

Best wishes,

Jason

Dear Marianne,

Jason and Jaroslav have given very nice suggestions regarding the same like acclimation in new media and various attachment substrates. I am also few months old in iPSC/ESC culture but got chance to deal with lots of cells at same time and have gone through such problems. Can you please tell me the magnification at which the colony picture was taken? Though I am not an expert, but that seems a differentiated colony (please correct me if I am wrong in that). Undifferentiated colonies generally look transparent and it's hard to distinguish individual cells in a perfect colony.

I encountered same problem where two stocks didn't attach at all with mTESR, then I saw that media was expired. Its a minor thing but please have a look that your media is within expiry date. Are any of your other iPSC/ESC lines working fine in the same media?

Matrigel is generally stable for long time after coating while growing cells, even I have grown ESCs on MG even for 30 days and colonies didn't come out. For coating, we generally incubate MG overnight at 4 degree and never had any problem with the same. You can also try if that works for you. I generally dissolve 250ul MG into 9.75ml DMEM:HAM's F12 and it works great.

One most important parameter regulating their growth is media change. I have seen that these cells can't tolerate even a one day delay in media change and get differentiate very fast. I have encountered this problem before and when I was strict with changing media, everything went fine. You can also consider this.

Further, adding FGF2 can help to increase the cell growth and proliferation. We observed this in our system when we were using 8ul/ml of stembeads which release FGF2 at a steady rate for 72 hours and the growth was so robust that it was pain to passage cells so fast. then we reduced that to 4ul and that worked. You can also try this as that might be an important factor. Though its a costly affair but worth risking for to save a cell line.

Hope it helps!

Hi,

The quality of iPS cells colony is very good. Try to enhance the density of iPS cells culture.

Good luck!

When you only have a sparse population of a particular iPSc clone, one thing that you can do is to use filtered conditioned media from a more dense culture. This will accelerate the growth of the sparse culture. In general this should not be necessary but it is a viable option.

Regarding matrigel, I'm continually surprised by people who use a particular dilution ratio without any consideration for protein concentration. In the instance of your "use 11.64ul of matrigel per 1ml of media," it is clear that you were given a concentration specific recommendation specific to a particular lot of matrigel.