One Cell, One Drop, One Click: Hybrid Microfluidics for Mammalian Single Cell Isolation

Abstract

Generating a stable knockout cell line is a complex process that can take several months to complete. In this work, a microfluidic method that is capable of isolating single cells in droplets, selecting successful edited clones, and expansion of these isoclones is introduced. Using a hybrid microfluidics method, droplets in channels can be individually addressed using a co‐planar electrode system. In the hybrid microfluidics device, it is shown that single cells can be trapped and subsequently encapsulate them on demand into pL‐sized droplets. Furthermore, droplets containing single cells are either released, kept in the traps, or merged with other droplets by the application of an electric potential to the electrodes that is actuated through an in‐house user interface. This high precision control is used to successfully sort and recover single isoclones to establish monoclonal cell lines, which is demonstrated with a heterozygous NCI‐H1299 lung squamous cell population resulting from loss‐of‐function eGFP and RAF1 gene knockout transfections.

Digital Microfluidics Chips for the Execution and Real-Time Monitoring of Multiple Ribozymatic Cleavage Reactions

“…The automation system was composed of an in-house Python 2.7 software to control an Arduino Uno microcontroller (Adafruit) driven control board. A 15 kHz sine wave output from a function generator (Agilent Technologies) was amplified by a PZD-700A amplifier (Trek Inc.).…”

Single-cell droplet microfluidics for biomedical applications

“…Depending on whether an external force is needed, droplet generation typically includes two types, that is, passive and active generation of microdroplets, as shown in Table 1. 58,[63][64][65][66][67][68][69][70][71][72][73][74][75][76] Passive methods require no external force, and have simple structure of the microfluidic chip. The formation of droplets is controlled mainly by modifying the microchannel structure and two-phase velocity ratio.…”

“…single-cell isolation. 70 Unfortunately, this system can only achieve the operation of a dozen droplets/single cells and its throughput needs to improve. The use of magnetic fields to generate droplets has the advantages of non-heat generation, operational simplicity, and avoidance of contact with samples.…”

“…Furthermore, Samlali et al developed a hybrid microfluidic platform based on a co-planar electrode component to precisely control the droplet generation for single-cell isolation. 70 Unfortunately, this system can only achieve the operation of a dozen droplets/single cells and its throughput needs to improve.…”

Recent advances in microfluidic devices for single-cell cultivation: methods and applications

“…58,161 The viability and growth of encapsulated single cells are influenced by droplet size and applied force during droplet formation. 220,221 Even though microfluidic devices allow multiple single-cell analyses in a few tens of minutes to avoid significant cell damage in a droplet, 222 longer cultivation typically requires additional care to maintain a favorable environment for single cells in droplets. 141 Based on these disadvantages from each structure, developing new structures capable of integrating with systems within microfluidic devices, which allows simple, high-throughput, and high-efficiency single-cell encapsulation, is highly needed.…”