Single-cell droplet encapsulation for drug exploration: A review of principles, methodologies, and future directions

• Droplet microfluidics technology enables over 90% cell encapsulation in high-throughput drug screening. • Hydrogel-based encapsulation creates biomimetic 3D microenvironments for single-cell analysis. • Integrated microfluidic platforms accelerate the design of targeted drug delivery systems at single-cell resolution. The differences in drug resistance and individualized efficacy caused by cell heterogeneity have become the core bottleneck in the process of drug development. Traditional population analysis can only obtain the average signal of cells, which cannot characterize true differences among single cells and masks cell heterogeneity. To overcome this bottleneck, single-cell droplet encapsulation technology has emerged. This technology captures tumor cell heterogeneity at the single-cell level, thereby accurately identifying drug-resistant subpopulations, accelerating drug screening, and guiding personalized therapy. Here, we provide an overview of single-cell droplet encapsulation technology, covering its fundamental principles, microfluidic droplet generation, cell encapsulation strategies, and downstream sorting methodologies. We further discuss its transformative applications in drug discovery, including: (i) target identification through molecular profiling of rare drug-resistant subclones, (ii) high-throughput screening for single-cell drug sensitivity assessment, and (iii) precision drug delivery via tailored strategies for specific cellular subsets. These advances aim to accelerate personalized medicine and expand the utility of droplet-based platforms in pharmaceutical research.