Method Profile
Measure apoptosis over time in individual ovarian tumor organoids exposed to iPSC-derived NK cells.
Resolve variation within a culture that bulk killing measurements can obscure.
Two ovarian cancer PDO lines from two patients: a high-grade serous omental metastasis and an ovarian mucinous carcinoma. NK cells came from one iPSC line.
Organoid/NK co-culture in 1:1 mixed media with 5% BME. Twenty NK cells were added per organoid unit, with imaging every 15 minutes for up to 11 hours.
Co-culture
1,500 organoid units and 30,000 iNK cells per well, with parallel organoid-only controls.
Imaging
Orange-labeled organoids, blue-labeled NK cells and caspase-3/7 activation; automated segmentation measures apoptotic area per organoid.
Fit a two-component Bayesian Beta mixture to endpoint apoptotic area. Apply an 8.6% threshold over time and a Gompertz model to estimate a persisting non-apoptotic fraction.
Media compatibility, immune killing and within-line heterogeneity.
Two PDO lines; three independent imaging experiments per line; more than 500 individual organoids per condition. NK identity characterization included 10 differentiation rounds from one iPSC line.
Organoid monoculture; pure NK or organoid medium versus mixed medium. Fresh and frozen NK function against K562 was assessed separately.
More than 75% of organoids displayed apoptotic events by 5 hours. Gompertz modeling estimated a 6.2% non-apoptotic fraction (credible interval 4.9–7.5%), despite observed NK engagement with some low-signal organoids.
Imaging hundreds of individual organoids revealed heterogeneous apoptosis after exposure to iPSC-derived NK cells. A small fraction remained non-apoptotic during observation, including organoids contacted by NK cells.
