High-throughput single-cell quantification using simple microwell-based cell docking and programmable time-course live-cell imaging

Abstract

Extracting single-cell information during cellular responses to external signals in a high-throughput manner is an essential step for quantitative single-cell analyses. Here, we have developed a simple yet robust microfluidic platform for measuring time-course single-cell response on a large scale. Our method combines a simple microwell-based cell docking process inside a patterned microfluidic channel, with programmable time-course live-cell imaging and software-aided fluorescent image processing. The budding yeast, Saccharomyces cerevisiae (S. cerevisiae), cells were individually captured in microwells by multiple sweeping processes, in which a cell-containing solution plug was actively migrating back and forth several times by a finger-pressure induced receding meniscus. To optimize cell docking efficiency while minimizing unnecessary flooding in subsequent steps, circular microwells of various channel dimensions (4-24 µm diameter, 8 µm depth) along with different densities of cell solution (1.5-6.0 × 10(9) cells per mL) were tested. It was found that the microwells of 8 µm diameter and 8 µm depth allowed for an optimal docking efficiency (>90%) without notable flooding issues. For quantitative single-cell analysis, time-course (time interval 15 minute, for 2 hours) fluorescent images of the cells stimulated by mating pheromone were captured using computerized fluorescence microscope and the captured images were processed using a commercially available image processing software. Here, real-time cellular responses of the mating MAPK pathway were monitored at various concentrations (1 nM-100 µM) of mating pheromone at single-cell resolution, revealing that individual cells in the population showed non-uniform signaling response kinetics.

Label and Label-Free Detection Techniques for Protein Microarrays

“…In each microarray, also known as ‘biochips’, thousands of reaction spots (capturing agent spots) are arrayed onto a few square centimeters. It is a remarkably versatile and useful platform, particularly when dealing with very limited sample volumes in genomics [1], proteomics [2,3,4], transcriptomics [5,6], glycomics [7,8,9], metabolomics [10], and cellomics [11] research. Partially automated dispensing systems, condition controlling systems and automated signal readouts enable sophisticated experiments to be carried out by non-specialized technicians.…”

Research progress of biomimetic superhydrophobic surface characteristics, fabrication, and application

“…However, the use of selective hydrophobic/hydrophilic surface to control liquid flow is an efficient control method. In this case, the micro/ nanostructure roughness of the microchannel can determine the liquid flow in a predetermined path, [38][39][40][41][42] as shown in Figure 16.…”

Microfluidic Bioreactors for Cell Culturing: A Review

“…Han et al, [288] reported a novel integrated microwell-structured microfluidic device that is capable of trapping single oocytes, fertilisation and subsequent embryo culturing. The device in an array format was used to capture and hold individual oocytes during the flow-through process of oocyte Integration of cell impedance analysis into a single-cell trapping microfluidic structure HeLa Impedance [277] Handling of cells in microfluidic platform using dielectrophoresis methods S.cerevisiae and sheep red blood [278] Continuous differential impedance analysis of single cells held by a hydrodynamic cell trapping HeLa Impedance [279] Determination of hydrogen peroxide (H2O 2) in individual HepG2 cells HepG2 Fluorescence [280] Single cell quantification using microwell-based docking and programmable live cell imaging S. cerevisiae Fluorescence [281] On-chip low power piezoelectric actuated micro-sorter for deflecting single particles and cells at high-speed.…”