High Throughput Single-cell and Multiple-cell Micro-encapsulation

Abstract

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one-and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 μm polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed.

Inertial Focusing in Microfluidics

“…The high speed of particles in inertial focusing flows is also tailor-made for single-cell encapsulation technologies, which are reviewed elsewhere (113). By matching the frequency of cell ordering in the longitudinal direction and the production of droplets, these technologies drastically improve the encapsulation efficiency of single cells (50, 114, 115). Examples of these technologies are highlighted in Figure 7 b , in which the production of droplets ( > 1,000 droplets per second) containing either single cells ( left ) or pairs of cells is imaged using a high-speed camera.…”

“…( a ) Measurement of individual cell deformability characteristics for disease detection at a rate of thousands of cells per second (108). ( b ) Coupling of inertial ordering of cells and droplet encapsulation, which drastically improves the number of droplets created that hold a single cell and allows controlled encapsulation of particle pairs (114, 115). ( c ) Improved viability during electroporation by taking advantage of the spinning and vortexing of cells trapped in expansion/contraction devices.…”

Microfluidic techniques for high throughput single cell analysis

“…Combining deterministic encapsulation[53] with precision biochemical microenvironment control and unmatched throughput, droplet-based segmentation has enabled a host of unique assays on the single cell level (Figure 2e). …”

“…d) Single cell hydrodynamic stretching for mechanical phenotyping[48]. e) Deterministic cell encapsulation for precise modulation of single cell environments[53]. …”

Jetting microfluidics with size-sorting capability for single-cell protease detection

“…The cells can be ordered along the microchannel under certain flow rates to form an equally spaced cell train before encapsulation into droplets. However, these methods require extremely high cell densities ( $10 7 cells/mL) to achieve effective cell encapsulation and to avoid the generation of a large number of empty droplets, limiting the application of these methods in assaying clinical samples with low cell abundance (Lagus and Edd, 2011). Moreover, hydrodynamic droplet sorting has been demonstrated to improve single-cell encapsulation rate with high input cell concentration ( $ 10 7 cells/mL) (Chabert and Viovy, 2008).…”