A Scalable Droplet Microfluidic Platform That Overcomes Large-Cell Constraints for Long-Term Plant Cell Culture and Embryogenesis Analysis.

Microspore-derived embryogenesis (MDE) is essential for doubled haploid (DH) plant breeding, but conventional bulk culture systems are limited by poor scalability and difficulty for automation. Adapting droplet microfluidics for plant cell culture is promising but challenging due to the large size and fragility of plant cells involved in embryogenesis, such as maize microspores (60-85 µm) and their expanding embryo-like structures (200-500 µm). Here, we present a droplet microfluidic platform engineered for large plant cells, integrating a tubing-based loading strategy that minimizes sedimentation losses, robust generation of stable 20 and 200 nL droplets optimized for large-cell encapsulation, and scalable 3D reservoir incubation for long-term culture. Optimized flow conditions and surface treatments enabled cell encapsulation with high fidelity, sustaining viability for over two weeks. Multi-modal viability assessment using fluorescence spectroscopy and microscopy (bright-field and fluorescence) confirmed that droplet culture preserves cell health comparably to bulk methods. The platform supports both single-cell and few-cell encapsulation modes, and increasing droplet volume and cell loading density further enhances embryogenesis rates and total embryo yield, respectively. Together, these advances establish a scalable, modular droplet microfluidic platform for high-throughput embryogenesis analysis and embryo production, offering new opportunities in plant developmental biology, precision breeding, and doubled-haploid technology.