Double resonant piezoelectric cytometry enables label-free mechanophenotyping of PRPF8-Mutant iRPE cells

Abstract Background Pathogenic PRPF8 variants cause severe retinal degeneration, but their biomechanical correlates in the retinal pigment epithelium (RPE) remain poorly characterized. We developed and applied a reconfigured double resonant piezoelectric cytometry (DRPC) platform for label-free mechanophenotyping of patient-derived induced pluripotent stem cell-derived RPE monolayers (iPSC-RPE, iRPE). Results The DRPC platform enabled real-time estimation of traction-associated surface lateral stress, storage modulus, loss modulus, and loss tangent during monolayer adhesion and maturation. Compared with control iRPE, the patient-derived line carrying the PRPF8 c.5792 C > T variant showed impaired barrier function, downregulation of selected cytoskeletal and contractility-associated genes, a descriptively delayed transition from negative protrusive to positive contractile surface stress, less stable maintenance of positive contractile loading, and different group-mean elastic and dissipative trajectories. These mechanical patterns co-occurred with qualitative differences in F-actin and vinculin organization and reduced integrin α5β1 fluorescence intensity. Conclusions These findings identify exploratory, variant-associated differences in dynamic mechanical response patterns in the studied patient-derived iRPE line ( PRPF8 c.5792 C > T) and support DRPC as a complementary label-free approach for monolayer-scale mechanophenotyping in inherited retinal disease models.