High precision, high throughput generation of droplets containing single cells

Abstract The generation of single-cell biological assays has become a strategic need for fundamental and clinical studies. Droplet microfluidics being sensitive, high throughput, and low cost, has been widely adopted for single-cell assays. However, precise manipulation of single cells using droplet microfluidics is still limited by the intrinsic randomness during single cell encapsulation, known as the Poisson limitation. It results in ambiguous single-cell assays with considerable cell losses. Here we present a novel cell-triggered splitting (CTS) method for deterministic and passive single cell encapsulation into droplets. We demonstrated the high efficacy (negligible cell loss) and high specificity (high cell loading percentage) of this method. With a simple working principle, this passive and label-free method reaches an unprecedented throughput of more than 3k Hz for droplet sorting, corresponding to 22,000 cell-loaded droplets per minute with less than 3% false events (empty droplets or doublets), a 100-fold improvement compared to existing label-free active droplet sorting methods. This asynchronous method requires no flow tuning and provides a general solution to different application scenarios, from high throughput encapsulation to manipulation of small cell populations. This method also applies to the encapsulation of other deformable particles such as hydrogel beads. The novel versatile tool may impact applications in single-cell sequencing, cell-cell/cell-bead interaction, and rare cell isolation. This passive and deterministic single-cell encapsulation method provides a practical solution to overcome the long-existing difficulty associated with the Poisson limit.