I am currently conducting my PhD research aimed at isolating and culturing cells from breast cancer tissues for further molecular and cellular analyses. Effective cell extraction from tissue samples is a critical initial step in this process, as it directly impacts the quality and viability of the isolated cells. Various enzymatic and mechanical methods have been employed for tissue dissociation, each with its advantages and limitations. However, the optimal protocol for extracting cells from breast cancer tissues remains a topic of interest and discussion in the scientific community. I am seeking insights, recommendations, and protocols from fellow researchers who have experience in this area. Specifically, I am interested in knowing which extraction protocol has been found to be the most efficient and reliable for isolating viable cells from breast cancer tissues. Additionally, I would like to inquire about the preferred enzyme or mechanical procedure used for tissue dissociation in these protocols. Your contributions will be invaluable in guiding my research towards the development of robust cell isolation techniques for breast cancer studies, ultimately contributing to advancements in our understanding of this disease and the development of potential therapeutic strategies."
Hello Amalia Kotsifaki,
The best approach would be to use a mixture of collagenase and hyaluronidase considering the composition of the breast tissue. Another important point to note is that slow digestion will give higher number of viable cells providing less harmful conditions compared to fast digestion.
I suggest you carry out an overnight digestion. After washing the tumor tissue with 1X PBS, you may cut the tissue
using surgical scalpel. The tissue fragments may be washed again with PBS, inserted into a 50 ml falcon tube containing the mixed enzyme solution ( 2mg/ml collagenase type I, 0.5mg/ml hyaluronidase and 0.1%trypsin dissolved in basal medium) and then incubated 16 hours, 300 rpm, 37°C, 5% CO2. After incubation you may fiter the homogenate using 100 μm cell strainer and then centrifuged at 1600 rpm for 10 min. Supernatant may be discarded and the pellet washed with 1X PBS and centrifuged at 1600 rpm for 5 min. The pellet may be resuspended with the complete growth medium. The cell suspension may then be incubated at 37°C, 5% CO2.
Use of mechanical disruption methods may be too aggressive and harmful for primary cells.
You may also refer to the article attached below for some more information.
https://cancerci.biomedcentral.com/articles/10.1186/s12935-023-03190-4#:~:text=Cell%20isolation%20with%20collagenase%20and,concentrations%20of%20collagenase%20and%20hyaluronidase.
Best.
Malcolm Nobre What would you consider the most effective and preferable method for cryopreserving these cells to ensure their viability and integrity for future experiments, given your extensive knowledge in this area? And medium preparation?
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