I am new to the organoid work and any suggestions would be helpful. I want to treat my organoid with different compounds and look for many readouts (proliferation, invasion, viability, EMT, chemosensitivity etc). Since these organoids are 3D and in matrigel, the assays are not as straightforward. I have a few concerns:
1. How to optimize the organoid size/diameter that I want to work with? Once optimized, how to make sure the organoid size/number is consistent across all the wells in an experiment?
2. For the plate reader based assays, can I normalize organoid number by protein content (Bradford), or nuclei (Hoechst)?
3. When I do assays with PI/Hoechst staining in a plate reader, how do I quantify these stainings in all the layers of the organoid? should I use z-stack in confocal?
4.Which is better - growing organoid in matrigel dots vs free floating matrigel?
5.Is isolating mRNA from organoid a straightforward approach or do I have to consider something?
Thank you!
hi Shilpa Patil
I do not work with Organoids but works with 3D cancer stem cells spheroids.
So, you can consider this, just a discussion.
1. In spheroid, single-single cells aggregates and form spheroid, so the number of cells seeded matter. Once it starts forming spheroids, the number of days it is cultured in the plate determines its size, since it grows. There are papers that demonstrated the diameter of spheroids & respective applications. So, I think in your case also numbers of cells and incubation period will have a role.
2. Well, not all plate reader assays need normalization. if it is required, you can go with normalization accordingly.
5. I think, for mRNA, you can directly go for isolation after collecting the organoids.
Regards
Saurabh
As you are testing compounds you could seed a plate (in the most appropriate way for your organoids) and then simply size and count them. You would to make sure the seeding was the same across groups. This also might remove the need for a plate reader and any normalization.
Simply counting and sizing of these 3D objects seems to becoming more of a standard assay in this area and follows what has been done in cancer research forever with clonogenic assays. Depending on number of plates and how you need to count you may want to consider automating that process (if possible) as it can be time consuming to do.
Here are a few hopefully relevant articles that may help with your questions above:
Article Attenuation of SRC Kinase Activity Augments PARP Inhibitor–m...
Article RON signalling promotes therapeutic resistance in ESR1 mutan...
Article Heterogeneity and dynamics of active Kras-induced dysplastic...
And an article on automation of this:
https://www.oxford-optronix.com/resources/quantifying-organoid-size-and-counts
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