Background

Cellular heterogeneity within a large cell population is common. Individual cell analysis will provide more accurate information about the cell-to-cell variation masked by the stochastic average in bulk analysis. Single cell culture, which is the first step of individual cell analysis, becomes very important.

Flow cytometry is a commonly used method for single cell analysis. However, the requisition of large cell numbers (more than 10,000) hurdled its applications in the research of limited precious samples. With the merits of low sample consumption, low analysis cost, microfluidics has been pursued by many scientists, especially in the research field of limited sample acquisition and expensive reagents needed, such as cell-based drug screening. Digital microfluidics, which utilizes electric signal to manipulate individual droplet has shown its promising in cell-culture related research. However, it is difficult to realize single cell capture on a flat electrode on digital microfluidics considering several hundred nanoliter sized droplets. Although several researchers reported that they can isolate single cell on digital microfluidics, the drawbacks hindered their further applications. For example, Gidrol’s group realized single cell isolation by adjusting the concentrations of cell suspensions (Rival et al., 2014). However, the single-cell isolation efficiency was quite low. Lammertyn’s group fabricated a series of micro sized hydrophilic micropatches on an electrode for single cell trapping (Witters et al., 2011). However, the multiple hydrophilic patches resulted in higher droplet transportation voltage, which will not only shorten the chip lifetime, but also cause potential damage to cells.

In our recent work, we developed a single cell culture method for primary tumor drug screening based on microfabrication and digital microfluidic technologies (Zhai et al., 2020). In the work, 3D microstructures were engineered on digital microfluidic chips for single-cell isolation and long-time culture. With novel combination of medium oil and additives, the droplet actuation voltage was lowered to 36 V, 4 times lower than normally used. Following, breast cancer drug screening was run on-chip with the designed microstructures showing consistent results as in conventional 96-well plates.

In the present protocol, we provide detailed steps for this single cell trapping method, including experimental details to perform single cell culture with the digital microfluidic system.