High-efficiency single cell encapsulation and size selective capture of cells in picoliter droplets based on hydrodynamic micro-vortices

Abstract

Single cell analysis has emerged as a paradigm shift in cell biology to understand the heterogeneity of individual cells in a clone for pathological interrogation. Microfluidic droplet technology is a compelling platform to perform single cell analysis by encapsulating single cells inside picoliter-nanoliter (pL-nL) volume droplets. However, one of the primary challenges for droplet based single cell assays is single cell encapsulation in droplets, currently achieved either randomly, dictated by Poisson statistics, or by hydrodynamic techniques. In this paper, we present an interfacial hydrodynamic technique which initially traps the cells in micro-vortices, and later releases them one-to-one into the droplets, controlled by the width of the outer streamline that separates the vortex from the flow through the streaming passage adjacent to the aqueous-oil interface (d). One-to-one encapsulation is achieved at a d equal to the radius of the cell, whereas complete trapping of the cells is realized at a d smaller than the radius of the cell. The unique feature of this technique is that it can perform 1. high efficiency single cell encapsulations and 2. size-selective capturing of cells, at low cell loading densities. Here we demonstrate these two capabilities with a 50% single cell encapsulation efficiency and size selective separation of platelets, RBCs and WBCs from a 10× diluted blood sample (WBC capture efficiency at 70%). The results suggest a passive, hydrodynamic micro-vortex based technique capable of performing high-efficiency single cell encapsulation for cell based assays.

Asynchronous generation of oil droplets using a microfluidic flow focusing system

“…For example, in our case, the inlet pressure equals the internal pressure of the balloon and the outlet pressure equals the ambient pressure, providing . The suitability of pressure pump-driven systems for the continuous generation of droplets has been demonstrated in several works 33–36 .…”

Lipoplex‐Mediated Single‐Cell Transfection via Droplet Microfluidics

“…Due to the limitation imposed by Poisson statistics for random cell loading, the single‐cell encapsulation efficiency of our platform was ≈18%. A higher single‐cell loading efficiency could be achieved if our platform is coupled with inertial‐microfluidic cell ordering with a curved channel, inertial‐microfluidic cell focusing in a long, high‐aspect‐ratio microchannel, or one‐cell‐to‐one‐droplet releasing by the hydrodynamic microvortices at the droplet pinch‐off interface …”

“…A higher single-cell loading efficiency could be achieved if our platform is coupled with inertial-microfluidic cell ordering with a curved channel, [33] inertial-microfluidic cell focusing in a long, high-aspect-ratio microchannel, [34] or one-cell-to-onedroplet releasing by the hydrodynamic microvortices at the droplet pinch-off interface. [35] The cationic lipid we used here is Lipofectin, a widely adopted nonviral vector for mammalian cell transfection, which consists of a mixture of positively charged lipid N-(1-(2,3-dioleyloxy)propyl)-n,n,n-trimethylammonium chloride (DOTMA), and helper lipid dioleoyl-phophotidylethanolamine (DOPE) at a 1:1 (w/w) ratio. [36] Cationic lipids form lipoplexes spontaneously with polyanionic nucleic acids upon electrostatic interaction, and the resulting complexes interact with the cell membrane and are internalized through endocytosis.…”

A Microfluidic Strategy for Controllable Generation of Water‐in‐Water Droplets as Biocompatible Microcarriers

“…The isolated compartments with tiny volumes and large specific surface areas are ideal microreactors, transporters, and mixers for microscale experiments . Droplet microfluidic offers a large number of applications in small molecule detection, drug screening, single‐cell analysis, functional microparticles fabrication, tissue engineering, etc.…”