An Endothelial Layer Changes Doxorubicin Responses in an Isogenic Heart Chip
A heart chip combined cardiomyocytes, cardiac fibroblasts and endothelium from one human iPSC line. Endothelial-containing cultures preserved beating and sarcomere organization during 1 μM doxorubicin exposure, while isoproterenol increased beating similarly with or without endothelium.
One Cell Line Supplied Three Cardiac Compartments
A study led by the Bellvitge Biomedical Research Institute examined how an endothelial interface changes drug responses in a human heart-on-a-chip. Published in Advanced Healthcare Materials, it used cardiomyocytes, cardiac fibroblasts and endothelial cells differentiated from the same induced pluripotent stem cell line. Comparing the complete tri-culture with cardiomyocyte–fibroblast cultures allowed the researchers to investigate the endothelial contribution while keeping the genetic background constant.
The device housed four membrane-based chambers, each with a 0.32 cm² central culture area. Cardiomyocytes and fibroblasts occupied one membrane face, and an endothelial monolayer occupied the other. Both channels were perfused from a shared medium reservoir at 5 μL/min. Drugs entered through the lower channel. This arrangement introduced a cellular interface and paracrine communication into the exposure experiment, rather than simply mixing all cells in a well.
The lineages required separate quality checks. Cardiomyocytes exceeded 95% purity by troponin T staining, while purified endothelial cells reached 98% and formed junctions and tube-like structures. Cardiac fibroblast identity was assessed across passages using several markers. Later passage caused drift toward a less cardiac-specific phenotype, so the investigators used an intermediate passage range for subsequent work.
Culture Choices Affected Baseline Function
Polyethylene terephthalate membranes supported cell attachment better than polycarbonate membranes, where the cells detached. A 90:10 cardiomyocyte-to-fibroblast ratio supported stable beating, whereas a 50:50 ratio allowed fibroblast overgrowth and impaired cardiac activity. Fibronectin supported endothelial maintenance during perfusion more effectively than gelatin, and endothelial growth medium was needed to sustain all three cell types.
After seven days of perfusion, the reported live/dead assay gave 91.2% viability for the cardiomyocyte–fibroblast compartment and 99.9% for endothelium. The tri-culture retained cardiac sarcomere staining and endothelial junctional organization. Endothelial tracer retention was comparable to that of human umbilical vein endothelial controls in the separate barrier assay. These observations established a functional baseline for the subsequent acute drug comparisons, rather than a chronic adult-heart phenotype.
Isoproterenol and Doxorubicin Produced Different Comparisons
Isoproterenol, a β-adrenergic agonist, was tested at 1 μM for one hour. Beating rate increased in both cultures with and without endothelium. The investigators found no significant differences in the assessed contraction duration, relaxation time or time to peak. Thus, the endothelial layer did not attenuate this chronotropic response under the tested delivery conditions.
Doxorubicin was first assessed in a separate cardiomyocyte viability experiment over 0.01–1,000 μM for 24 hours, yielding an IC50 of 8.7 μM. The chip comparison then used 1 μM doxorubicin for 24 hours. Without endothelium, beating rate fell by 72%, and sarcomeres shortened and became disorganized. With endothelium, cardiomyocyte beating and sarcomere organization were substantially preserved.
The functional comparison was repeated in three independent experiments, with five measurements in the cardiomyocyte–fibroblast group and six in the tri-culture. Sarcomere measurements had their own sampling scheme. These repetitions came from the same iPSC background. The study therefore demonstrates a within-model effect of cellular composition, rather than variation across patient genotypes.
Preserved Cardiac Function Did Not Identify the Protective Mechanism
Doxorubicin disrupted endothelial VE-cadherin organization even when cardiac function was preserved. Baseline sarcomeres were also longer in tri-culture than in endothelial-free cultures. These findings leave several explanations open: changes in drug delivery, endothelial signaling, fibroblast interactions or differences in cardiomyocyte maturation could contribute. Drug concentrations on each side and individual candidate protective signals were not directly resolved by the comparison.
The result shows that a model’s cellular composition can change an acute cardiotoxicity readout in a drug-dependent way. Immature iPSC-derived cells, a seven-day maintenance period and one doxorubicin exposure condition define the comparison’s current scope. The matched bi- and tri-culture configurations provide a system for separating exposure effects from multicellular mechanisms in subsequent drug-safety studies.