We have a problem during breast cancer organoid passaging. we can not disrupt compact structure of organoid. We tried trypsin (4-6min 37 C + pipeting ) but organoids disscociate partially. Then we tried trypLE (10 min 37C) but it also disscociate organoids partially. Can you help me with breast cancer organoid passaging?
Hello Merve,
Depending on the resiliency of the breast cancer cells used in your study, you may try more vigorous pipetting or vortexing for better mechanical disruption or increasing incubation time with trypsin (~15 min) o trypLE (~20 min).
If these suggestions are still not working, please, see below some protocols and reagents that may work.
-ATCC general protocol: https://www.atcc.org/resources/culture-guides/organoid-culture-guide#:~:text=Organoids%20are%20passaged%20with%20a,or%20recovery%20will%20be%20impaired.
-MD Anderson Cancer Center protocol: https://www.mdanderson.org/documents/Labs/Taniguchi%20Laboratory/Organoid%20Passaging.pdf
-Check out the organoid dissociation in this manuscript (page 3): Article Breast cancer organoids from a patient with giant papillary ...
-Some recommendations from 10xGenomics to obtain single cell suspension from organoids (if that's what you are going for): https://kb.10xgenomics.com/hc/en-us/articles/360058716812-Are-there-any-recommendations-for-working-with-organoid-tissue-for-10x-single-cell-assays
-Another reagent to consider: https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/351/666/scm300ds.pdf
Hope this helps. Good luck!
Use Corning Cell Recovery Solution or Cultrex Organoid Harvesting Solution
Breast cancer organoids can be dissociated with trypLE, but the ease of dissociation may vary depending on factors such as organoid density, ECM composition, and trypLE concentration. Optimization of the protocol may be necessary for efficient dissociation.
Hi Merve,
During my Master's, I worked with tumor spheroids from breast cancer and faced some difficulties when trying to dissociate them.
One thing that worked for me was incubating the cells with StemPro™ Accutase™ Cell Dissociation Reagent for 10 minutes. After the incubation, I performed reverse pipetting at least 5 times and successfully dissociated the cells. I hope this information is helpful to you.
Are the breast cancer spheroids being held together by an externally added matrix?
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