Hello.
I am growing human fibroblasts.
Spread 20,000 cells on a 12well plate and spread them.
When I was observing how fast the cell saturation was, the border was formed on the 5th day, so the lower part seemed to dry, twist and die. The culture medium was good enough.
Why does this happen?
thank you.
if the confluence reaches 100% and the cells start to squeeze together this negatively affects their viability and after a while they start to die. Possibly they were confluent for a day or two and start to die or most of the cells do not adhere to the plate when passaged
Thrombin promotes fibroblast mitosis, and thrombin starvation induces apoptosis. Ca+ is essential for thrombin activity. Either thrombin starvation or Ca+ depletion can induce apoptosis.
Diego Raffo
As you said, it seems that the cell is starting to die when the confluence is over 100%. thanks for the advice.
Lewis S Coleman
I will check to see if there is a Ca+ deficiency in the way I culture. I was the first to know that cell death due to Ca+ deficiency occurs, so I will refer to the attached file. thank you.
Hi, I guess confluency of your cells is too high, therefore they entered decline phase . You should passage cells when the confluency is approximately 80%. Also the microscope you use shows cells in 100x I guess. It is also an another parameter should be awared of. You can observe confluency in 4x or 10 x better to see a bigger image of the cells. This will help you to observe confluency.
Nazlıcan Yürekli
It was my first time growing human fibroblasts on a low scale, so I didn't know they would die so quickly. Thanks for the advice!
Hi
I think that the problem lies mainly in the confluency and the number of cells seeded per Well.
Also, you must pay attention that the fibroblast expands as it grows in shape and their size will be large, but the number is less.
The conclusion is that compared to other cancerous lines, fibroblasts expand more in size than their number that make them die on the border.
Best regards
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