Modeling Antibody-Mediated Endothelial Activation in a Human Vascularized Heart Wall Tissueoid-on-Chip

Introduction: Antibody-mediated rejection (ABMR) remains a leading cause of cardiac allograft failure. While complement-dependent cytotoxic ABMR is well characterized, subclinical and non-cytotoxic donor-specific antibody (DSA)-mediated endothelial injury is increasingly recognized but poorly understood, largely due to the lack of physiologically relevant human models. We developed a human vascularized heart wall tissueoid-on-chip (HWToC) to model DSA-endothelial interactions in a cardiac microenvironment and evaluate the resulting injury phenotype.
Methods: HWToCs were generated by embedding primary human cardiomyocytes, cardiac fibroblasts, and endothelial cells within a fibrin-thrombin hydrogel in a custom PDMS microfluidic device. Endothelial cells were seeded into adjacent channels to promote vascularization under pressure gradient-driven perfusion. Tissueoids were maintained for 23 days and characterized by bright-field imaging, dextran and whole-blood perfusion, live/dead staining, and immunofluorescence for CD31, collagen I, cardiac troponin T (CTNT), HLA-I, and DAPI. DSA-negative (DSA-) and DSA-positive (DSA+) allosera, characterized by single-antigen bead assays for anti-HLA reactivity, C1q binding, and IgG3 subclass, were perfused at 1:100 dilution for 36 hours on Day 23. Post-exposure assessment included immunofluorescence for CD31, IgG, C4d, ICAM-1, and HLA-I.
Results: HWToCs demonstrated progressive tissue maturation over 23 days, with increasing organization of the CD31+ vascular network, collagen I deposition, and CTNT expression. Dextran perfusion confirmed functional vasculature, and whole blood perfusion demonstrated physiological flow compatibility.

DSA- serum exposure produced no detectable changes relative to control. DSA+ exposure induced slight cytotoxicity without fully compromising vascular network functionality, as confirmed by live/dead and dextran perfusion assays. Immunofluorescence revealed modest IgG binding and low-level C4d deposition in DSA+ chips, with preserved but subtly altered CD31 and collagen I patterns

Notably, the C4d deposition was markedly lower than in our previously reported liver tissueoid-on-chip model, consistent with the absence of local complement production by cardiac cells and the high serum dilution used. This phenotype resembles non-cytotoxic DSA-mediated endothelial activation rather than overt complement-dependent vascular destruction.
Conclusion: The HWToC platform recapitulates a subclinical, non-cytotoxic DSA injury phenotype characterized by endothelial activation, low-grade complement deposition, and preserved vascular architecture. The expression of autoantigens such as collagen I further positions this model as a tool for investigating the role of non-HLA antibodies in cardiac allograft injury. This platform complements ABMR models and provides a translational system for studying the spectrum of antibody-mediated cardiac allograft pathology.