This shape gradually spreads. Even if you install new cells again, it will change like this at some point. Has anyone experienced the same thing as me? I tried using Matrigel again by aliquot it. same.
Please help me.. Besides the solution, please let me know what methods you have tried when experiencing similar situations, or if you have seen something like this around you.
Hi,
It looks like the cells losing anchorage on the plate. Make sure you have diluted matrigel properly and the correct concentration is used. Check the expiry date and shelf life as well. Try coating the dishes with Vitronectin, which works better than matrigel for iPSCs.
Muhammad Nihad A S
Thank you for your answer!
If the cells lose their anchorage, is it irreversible? Even if i put cells with that morphology on an i Matrix-511 coating, they will grow in torn form and die..
And Matrigel is dissolved with the dilution factor listed in the CoA, referring to each lot number.
Is it possible to coat with fibronectin or geltrex instead of vitronectin?
If the cells lose anchorage, they will float and form embryoid body-like structures or may die off if they are not in contact with nearby cells. Usage of Rock inhibitor on the first day of passaging can prevent this cell death.
When I initially cultured iPSCs in a Geltrex (which was expired) coated plate, they didn't attach and formed these structures, as shown in the image below. When these 3D cells were replated in Vitronectin-coated dishes, the cells attached and formed good colonies. Since then, I have been using Vitronectin. I believe Geltrex will also work if it is a fresh one.
If you see floating colonies, collect them and replate them in a different matrix available in your lab.
I hope you are not pipetting onto the colonies when you are giving a media change that would also result in such morphologies.
Further considerations:
Make sure the media is not old (not more than 2 weeks), and both media and cells are at the same temperature when the media is changed (thaw the media at room temperature away from light, and keep the cells inside the LAF until it comes to room temperature before changing the media.)
Muhammad Nihad A S
Thank you for your attention to my problem
but maybe, my problem is not same attatched image ㅜㅜ
my iPSC or ESC morphology is changed that circle to crumble,, like net
already i have used Y237632(Rock inhibitor), but that is no effects,and used fresh matrigel not saw gelation
that problem is suddenly occur.. I did well iPSC maintain and differentiation to neurons before seen morphology
anyway, thank you for suggestion many variable situation!!
Ti Ti I faced a similar problem after I was able to grow and expand my iPSCs suddenly I was facing a similar problem.
If you figured out the problem please share your findings.
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