I work with ovarian cancer cell line organoids embedded in matrigel, they grow well the A2780 cells, but once I start using 10% formaldehyde to fix them they disintegrate...does anybody have a protocol for fixing the organoids??? how do you manipulate individual organoid blobs if you want to move them to a tube for instance?? do you scoop the organoid/matrigel blobs?? or do you aspirate them with a blue tip???
good question. Treat them as small pieces of tissue. Use small cryomolds 5x5mm or so, fix for 15-20 min in 4% fresh paraformaldehyde, then carefully remove PFA (put a black paper underneath to make sure you accidentally not remove the organoids, use 100ul pipet to move liquids in-out), wash 3 times with PBS, then add 10% sucrose made in PBS, then (in 15-30min) replace with 20% sucrose, then (in ~2h, when the organoids will start sinking, which means they are saturated with 20% sucrose) replace with 30% sucrose, keep in 30% sucrose until the organoids sink to the bottom (3-4 hours), then remove 3/4 or so of 30% sucrose, fill cryomold with OCT sol-n, prepare dry ice/ethanol bath and a bucket with dry ice, put cryomolds one by one carefully on top of dry ice/EtOH bath (the bottom should be inside , the top must not be covered with EtOH, use forceps), OCT will start changing color to whilte (=freezing), this takes about 30 seconds, when it is white -place into dry ice bucket and go to the next mold. If you do them all at once there is high chance EtOH will get on top of liquid OCT and OCT will start bubbling and bubbles will freeze -not good. Check our papers https://www.liebertpub.com/toc/scd/24/23, Article Transplantation of Human Embryonic Stem Cell-Derived Retinal...
,https://onlinelibrary.wiley.com/doi/abs/10.1002/term.3017?af=R, we used this method, it works great. Good question and good luck
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