I am super confused right now about the seeding density for fibroblast. I searched online and most of the company protocol said 4000/cm2. However, based on my own experience, that's clearly not the case. I then did a little bit more research and see people suggesting 10000-30000/cm2. Could anyone please explain these to me? (why ATCC for example suggest 4000/cm2?)
https://www.researchgate.net/post/Seeding-cells-in-different-sized-dishes-plates
The above discusses seeding density in different dishes, which may be helpful. It also depends on the experimental purpose, stages of passage, species and other factors. We used mice dermal fibroblasts- See below. Good luck.
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Shin Murakami Thanks for helping. Based on the chart in the link and all my previous experience. Seeding density should be around 10000-40000 cells/cm^2. So I am very curious why ATCC and Lonza (big cell company) all suggest 2500-5000/cm^2 for human lung fibroblast or fibroblast in general. I feel like it can't be a careless mistake, but I couldn't understand the reason behind it.
link for ATCC culture suggestion: https://www.atcc.org/products/pcs-201-013
Hello Yuhao Wan
2500-5000/cm^2 is suggested if you are not planning to make any sort of manipulations right after so that you avoid as much as possible trypsinizing and splitting your cells. Meaning that if you are not planning to do any transfections or drug treatments on the day of plating (or consecutive day of platting).
Based on my experience with fibroblasts (MRC5, CAFs, and Human primary prostate fibroblasts) I found that 100,000 cell/9.6cm2 (6- well plate) is the best if you are planning to do an experiment on the day of platting or on the consecutive day. And then for sure, you scale up and down based on your culture plate and the surface area you are using exactly.
I hope this was helpful for you
Good Luck
Lina
Hello Yuhao Wan,
In relation to what Lina Mesilmany pointed out, it depends on your specific aim and protocol. If you are working with viable but growth-arrested fibroblasts or your plan is to extract total protein/nucleic acids from them immediately, it might be convenient to culture the cells at a high density. Otherwise, if you are dealing with proliferating fibroblasts and are planning to culture them for several days/weeks, then it would be desirable to culture them at lower cell densities. Cell culture over confluence for an extended time causes major, and often undesirable, changes in cell metabolism.
Wishing you success,
Laura
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