Hi! Our lab just starts working with colorectal and breast cancer organoids and we faced a problem with passaging of organoids: there are much fewer organoids than before splitting.

Currently, we use such protocol: we scrape the entire surface of each well using the cell scraper or pipet tip that was cut, transfer everything from the well to the tube, centrifuge the tube at 500 × g for 3 minutes at RT, aspirate the medium without disturbing the gel (Matrigel), that contains organoids at the bottom of the tube, add 10 volumes of TrypLE Expres, pipet 5 times and then incubate for 12 minutes in the incubator (+37 °C), add 1/10 volume of FBS, pipet up and down three times, centrifuge the tube at 500 × g at RT for 3 minutes, aspirate supernatant. At this stage, we always resuspend the pellet in 100 µl of DMEM and count the number of organoids. We notice that there are ~4 times fewer organoids than before splitting.

Is it normal? What we are doing wrong? Could you please recommend another protocol for organoid splitting?

Thanks in advance!