Hello Soham Naskar

The choice of method depends on the type of research question. Each system has its own advantages and disadvantages, and you must carefully consider which system is best suited for your application.

Hanging drop method is the most common method. In the hanging-drop method, cells in the culture medium suspension are placed on the underside of petri dish lids. The cells accumulate at the tip of the drop, spontaneously aggregate, and form spheroids. However, this method is limited by the difficulty in exchanging the culture medium, thereby preventing cells from being cultured for more than 3 days. Furthermore, cells within the spheroids are subject to diffusive oxygen and nutrition supply. Additionally, it is critical to prevent the necrotic damage to the cells at the core of the spheroid and to control the size and composition of spheroids.

On the other hand, if you use sphere-formation assay, it will provide useful tool to assess the stem cell population residing in cancer cell lines. The advantage of using the sphere-formation in vitro assay is to isolate, propagate, purify, and amplify specific population of cancer stem cells. It enables studying stem cells at different stages of their formation (at different generations) and detecting markers of their signaling pathways. It also solely depends on the functional intrinsic property of stem cells in forming a complex structure in a 3D environment, namely self-renewal ability and differentiation potential.

In the sphere formation assay, cancer stem cells are isolated using anchorage-independent sphere culture. Cancer stem cells can grow on ultra-low attachment plates that are coated with a layer to inhibit the attachment of cells. When cells are grown in serum-free and non-adherent conditions, cancer stem cells can survive and clonally expand to form spheres, whereas differentiated tumor cells undergo apoptosis due to their anchorage dependence.

Below attached links will be helpful.

Article Establishment of Cancer Stem Cell Cultures from Human Conven...

Article Establishment and Characterization of a Human Small Cell Ost...

Preprint Isolation of cancer stem cells by sphere formation assay v1

Best.

Dear Soham Naskar ,

In addition to what Malcolm has already mentioned along with the valuable references, I would recommend to start growing cells in serum free DMEM supplemented with EGF and bFGF. We started with adherent C6 (rat glioma) and MCF-7 cells first in regular serum-containing DMEM with PenStrep, and then transitioned to a different in vitro microenvironment to stimulate cellular transitions to cancer stem cells (CSCs) or rather tumorsphere cultures, by using serum-free DMEM (+EGF, +bFGF, +PenStrep) that we referred to as "stem cell medium". The related publication is below.

Once the cells transition to CSCs, it is a good idea to maintain and grow them in ultra low attachment surfaces as Malcolm pointed out. Recently, we have been growing and maintaining cancer stem cells in DMEM/Ham's F-12 medium but with the same growth factors mentioned above and this works equally well. In future, you can also select CD24/CD44-high cells (FACS sorting) to more specifically select and enrich for cancer stem cell populations.

https://pubmed.ncbi.nlm.nih.gov/32816185/

Hope this helps. Please let me know if you have further queries.

Good luck!