Method Profile
Healthy-donor iPSC liver organoids are paired with drug-primed donor-matched T cells to connect HLA-associated immune activation with liver-cell injury.
Study donor-dependent adaptive immune drug injury while controlling allogeneic mismatch and direct compound toxicity.
Healthy blood-cell-donor iPSC liver organoids and autologous primary CD8 T cells. The priming cohort contains four HLA-B*57:01 carriers and four non-carriers; the injury experiment uses selected donor-matched pairs.
500 μm hydrogel microcavities with approximately 250 cells per aggregate; day 23 liver organoids receive 100 μM flucloxacillin for 72 hours before 72-hour coculture. Figure 5 specifies a 5:1 effector-to-target ratio; the methods instead state 1:1 and 1:5.
Matrix and monoculture controls
Initial enrichment medium contains 2% Matrigel despite matrix-free wording; no bulk dome embedding. Seven-day chlorpromazine, flucloxacillin and streptomycin tests with day 3 redosing; flucloxacillin up to 1 mM.
Immune comparisons
Drug-loaded dendritic-cell priming at 1,000 μM flucloxacillin, unprimed T cells, autologous/allogeneic pairing and anti-CD3/CD28 stimulation controls.
Labeled liver-cell death and cytokeratin 18 release are assessed alongside immune secretion. The four-carrier/four-non-carrier priming cohort and the inconsistently reported coculture donor series are distinct. Clinical sensitivity and individual clinical outcomes were not evaluated.
Direct-toxicant controls, donor-mismatch optimization and HLA-stratified priming followed by selected autologous injury assays.
Three iPSC lines in the array-format comparison; four carrier and four non-carrier priming donors; Figure 5 n = 5 assay measurements. The coculture Results initially report three carriers and three non-carriers, but subsequently describe two strong and two weak carrier responders.
Drug-free/unprimed, non-carrier, weak versus strong carrier, T-cell-free, allogeneic and autologous controls.
Flucloxacillin alone caused no direct injury up to 1 mM. Two strong carrier donors showed approximately fourfold liver-cell death with CK18/TNFα/granzyme B increases after primed-T-cell coculture; weak carriers/non-carriers lacked comparable injury. Strong nonspecific autologous stimulation could lower albumin without overt death.
Healthy-donor liver organoids tolerated flucloxacillin alone, while drug-primed autologous CD8 T cells caused injury in two strong HLA-B*57:01 responders. Weak carrier responses and non-carrier controls show why allele status and immune activation need separate measurements.
