Method Profile
Select a cardiac organoid formulation using health and functional measures, then compare drug-induced beating changes with viability.
Detect sublethal functional cardiotoxicity that may be obscured by residual beating in damaged monolayer cultures.
Human WTC11 calcium-reporter iPSC-derived mixed population, approximately 22.31% cardiomyocytes, 76.40% fibroblasts and 1.29% endothelial cells; 20,000- versus 80,000-cell aggregates.
Self-assembled suspension organoids followed at 2, 7 and 14 days; calcium-transient imaging and structural/health assays; selected 20,000-cell formulation versus 2D cultures with 48-hour doxorubicin at 0.1–10 μM.
Cardiac culture formulations
One WTC11 origin; two seeding densities and parallel monolayers. Low endothelial fraction does not establish vasculature.
Separate functional and viability readouts
Calcium-reporter beat-rate change relative to baseline; Calcein-AM/EthD-1 viability ratio; apoptosis, mitochondrial and gene-expression measures for model selection.
Fit separate 48-hour drug-response curves for viability and beating-rate change; calculate the selectivity index as viability IC50 divided by functional IC50. Most data are mean ± SEM; confidence intervals accompany drug-response estimates.
Density/time characterization followed by a one-drug comparison of function and viability in organoids and 2D cultures.
Day 7 apoptosis: n = 9 smaller and 10 larger organoids. Drug Figure 5: n = 15 2D measurements and 13 organoids per concentration across four biological replicates; one iPSC origin.
80,000-cell organoids for formulation selection; matched 2D cultures and vehicle for doxorubicin.
Day 7 apoptotic fractions were 0.075 versus 0.212 in smaller/larger organoids. Doxorubicin viability IC50 was 3.61 versus 0.69 μM in organoids/2D; functional IC50 was 0.84 versus 2.41 μM, with selectivity indices of 4.30 versus 0.29. Overall baseline beating measures did not significantly separate the density/time groups.
A human stem-cell-derived cardiac culture seeded with 20,000 cells had less apoptosis than the larger comparison at seven days. In subsequent doxorubicin tests, its beating response changed at lower concentrations than its viability, reversing the ordering observed in two-dimensional cultures.
