We are culturing hESCs using TeSR AOF kit and passaging with Accutase. The cells were in good condition, but one day after passaging the colonies suddenly formed weird shapes and every day there are a lot of cells dying.
It seems cells are being excluded from the colonies and died. Has anyone encountered such problems?
Hi there. This much cell death is usually observed due to improper passaging. I'd advice you to check your coating reagents/protocol and minimize agitation and timing during accutase treatment. The mESC-like domed-morphology might indicate another stressor, but I suspect there is a problem with the coating reagent you are using.
You may also consider adding before-after images of those cells during passaging and a detailed protocol you use (i.e. was there y-27632?), describing what exactly has been done to those cells throughout this process.
Hi Jasmine Ban
When culturing hESCs and observing abnormal colony shapes and cell death post-passaging, consider the following: Limit Accutase exposure time and ensure it's fresh and at the right temperature. Verify that cells are plated at the optimal density and that the TeSR AOF Kit is within its expiration date, stored correctly, and mixed well. Ensure a uniform surface coating with an appropriate matrix, check for microbial contamination, avoid rapid temperature changes or mechanical stress during pipetting, and maintain a consistent feeding schedule with regular media changes. Adjust based on these factors and monitor the cells closely.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line