Microfluidic platform for nanoliter qPCR of several retinal pigment epithelial (RPE) cells.

The clinical implant of stem cell-derived retinal pigment epithelium (RPE) monolayer for age-related macular degeneration requires rigorous validation of the monolayer's cellular maturity. Conventional bulk molecular assays lack the sensitivity to resolve cellular heterogeneity and are prone to damaging the RPE monolayer. This study developed and validated an automated nanoliter-scale microfluidic platform for single-cell transcriptomic quality control of engineered RPE monolayers. A compact polydimethylsiloxane -based microfluidic platform was developed to generate monodisperse 10 nL RPE cell cDNA droplets and 90 nL quantitative PCR (qPCR) reagent droplets using dual-focused-flow geometries. Deterministic droplet fusion was achieved via direct current electrocoalescence, enabling ∼100% merging efficiency. An auxiliary co-flow spacing mechanism was implemented to prevent secondary coalescence during downstream transport. Automated droplet collection into oil-filled 96-well plates was achieved through a custom two-dimensional gantry system, and droplet volume consistency was verified using a Python-based computer vision algorithm. Biological validation was conducted using H14 human embryonic stem cell-derived RPE cells targeting β-actin and lineage-specific markers MITF1, MITF2, PEDF, and PMEL17. The platform demonstrated stable generation and deterministic merging of nanoliter droplets, yielding uniform 100 nL reaction volumes suitable for qPCR analysis. Automated volumetric verification confirmed high droplet uniformity and reliability. Gene expression analysis revealed robust detection of housekeeping and lineage-specific genes at nanoliter scales. These findings demonstrated the feasibility of performing nanoliter-scale qPCR using the proposed microfluidic workflow. The workflow enabled reliable detection of extremely low quantities of nucleic acid using a widely available commercial qPCR platform, providing a practical and accessible approach for routine laboratory and translational research applications. The system-maintained assay sensitivity while significantly reducing reagent consumption and sample input. This automated microfluidic platform enabled reproducible, sample-efficient, single-cell transcriptomic validation of stem cell-derived RPE monolayers. By integrating droplet generation, deterministic merging, automated collection, and computational verification, the system addressed key limitations of bulk molecular quality control of RPE monolayers. This work established a scalable and cost-effective nanoliter qPCR framework for high-resolution molecular quality assurance of regenerative cell therapies.