I passaged my hiPSC into vitronetcin coatead plate with ROCK inhibitor in E8 medium. The 2nd day it looks good with about 40% confluency. But after I changed the medium to E8 without ROCK. I noticed under the microscope that the rim of the cells were detached. I changed the medium very slowly. But on the 3rd day when I checked them. A lot cells died and only a bit were still attached but in a rounded morphology.
Does anyone know why does the cell layer detach from the rim after changing the medium? Thank you very much!
Hi Ya,
Most likely your vitronectin coating fails to support cell attachment after removal of ROCKi. I had similar problem with Geltrex. Make sure your vitronectin aliquots are prepared correctly without exposure to warm temperatures at any stage and prepared with correct dilution rates. Also, make sure you coat plates at least for a few hours to overnight at 37oC but no longer than that as coating material will start degrading at high temperatures with extended times. If you coat plates too short (30min-60min), coating may not be complete. Another advice is once you thaw an aliquote, you should consume it in less than ~2 weeks. They usually don’t have long shelf life at 4oC once thawed. These should improve your cell attachment and survival. Good luck with your future experiments!
Hi Ya Gong ,
I agree with the points suggested by Emre Bektik .
If you have been following all the steps he recommended and still notice high cell death then maybe also check the E8 medium you are using. Does it contain the necessary supplements? Also the medium should not be too old or too cold when added to the cells. The medium stays good for about 14 days and not longer. In case you use water bath to warm your medium I could recommend to make small aliquots of the medium and use as much needed (instead of warming the entire medium bottle everyday).
Hope you are able to figure out better conditions for your iPSCs.
Good luck!
Best wishes
the plating density may be way too high. The cell passage may be too high (what is the cell passage you are using?). The cels could be over-treated with Ri (if this happened -they permanently change and it's hard to have nice & round pancake-like colonies, they become more like fibroblasts). Vitronectin is a very easy coating to make and its hard to make a mistake. I agree on coating and using coated dishes soon (in a week or so). It seems like the cells (the stock) may be a problem -obtain a vial with early passage cells (passage 22-25). Plate to have 20-30 colonies on p60 or 50-100 colonies on p100, allow to grow to 5-6 days, pick the best colonies manually, and create a bank of cells selected from best colonies. One has to do such selection once in a while unless you have an unlimited source of early-passage hESCs. If you are not sure about the passaging history of your hESCs (somebody gave them to you) then it's very hard to make any assumptions and conclusions (again -obtain another vial). I am happy to look at the images (please attach few images of your colonies), this will be most helpful
Few simple and routine suggestions;
1. Thaw a vial in 1 well of 6-well plate and then split once it's ready. Don't thaw frozen vial into multiple wells because most of the cells will die. Also, make sure you gently wash the thaw vial cells with 5ml of E8 media to remove freezing media and DMSO. After you thaw cells, change media only after 48hrs allowing cells to attach properly.
2. Make sure you use warm media to replace use over.
3. Make sure your frozen vial was not contaminated. If it was contaminated then the will cell attach for 24 hrs and then slowly start detaching with bacterial multiplication.
4. Don't make single-cell passages for hiPSC. A clump of 7-10 cells is highly recommended for better growth and attachment.
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