Hello Sanyam Jain

CFU-F assays often involve plating a small number of MSCs (e.g., 100-500 cells per plate. At low densities, MSCs might not produce enough of the soluble factors (like growth factors and cytokines) needed for their own survival and proliferation (paracrine signaling). On the other hand, in regular standard monolayer culture, higher seeding densities are used. In high density cultures, MSCs can interact with each other, releasing and responding to these factors and creating a more supportive environment.

MSCs need a suitable surface to adhere to and spread out, which is crucial for their survival and proliferation. If the surface area in the CFU-F assay is not optimized for cell spreading, MSCs might not be able to attach properly and could undergo apoptosis. In regular standard cultures, the larger surface area allows for better cell adhesion and spreading.

In terms of nutrients, in CFU-F assays, cells may quickly exhaust the available nutrients in the surrounding media, leading to starvation and cell death. Even if nutrients are available, the metabolic demands of MSCs during the early stages of colony formation might be higher than what's available at low densities, causing stress.

There are a few suggestions which you may want to consider.

1. You will need to optimize the seeding density. Instead of low seeding density try to adapt the CFU-F assay for higher densities. Using high cell densities in CFU-F assay is generally not recommended, but you can always optimize the seeding density to allow for colony formation without reaching confluence too quickly. At higher densities, colonies may merge, making it difficult to differentiate between individual colonies. So, closely monitor the culture to ensure that colonies are distinguishable from each other, even at higher densities. Your goal should be to find a balance between sufficient cell numbers and the ability to distinguish individual colonies for accurate counting.

2. CFU-F assays often require the addition of specific growth factors and cytokines to stimulate the proliferation and differentiation of colony-forming cells (CFCs). You may use conditioned media in CFU-F assay. Mesenchymal stem cells may be cultured in a suitable growth medium. After a period, the medium may be collected and filtered to remove the cells, resulting in conditioned medium. It contains secreted factors, like growth factors and cytokines.

3. Ensure you are using cells of passage number ≤ 2. The number of times a cell line has been passaged can affect its growth characteristics and colony-forming ability.

Best,

Check cell viability, optimize plating density, use correct media and plates, ensure proper incubation, extend culture time, and include a known MSC positive control.