Size-Based Sorting and In Situ Clonal Expansion of Single Cells Using Microfluidics

Isolating and analyzing individual cells from a mixed population is vital for understanding cellular heterogeneity and behavior. Prior research has shown that traditional cell culture methods often fail to maintain cell viability during long-term observation. This gap motivated the development of new tools for precise cell sorting and tracking. It was already known that cell size can vary significantly during the cell cycle, but linking size to specific phases remained unclear. No prior work had resolved how to integrate sorting with in situ clonal expansion. Existing methods lacked the ability to perform both tasks efficiently in a single device. This uncertainty drove the need for a system that could sort cells by size and monitor their growth over time. The challenge of maintaining cell integrity during fluid flow also remained unaddressed. This paper introduces a solution that combines filtration and pneumatic control to achieve these goals.

The aim of this study was to develop a microfluidic system capable of sorting cells by size and enabling in situ clonal expansion. The specific problem addressed was the difficulty in maintaining cell viability during long-term culture and imaging. The motivation came from the need for a reliable method to study cell lineage and growth kinetics. The system needed to avoid shear stress while allowing for accurate cell sorting. The researchers proposed integrating filtration channels with a pneumatic valve to control fluid dynamics. They also aimed to correlate cell size with cell cycle phases using time-lapse imaging. The study sought to evaluate how device parameters influence pressure drop and cell deformation. The ultimate goal was to create a platform for single-cell analysis in biomedical applications.

The researchers designed a microfluidic device with four filtration channels of varying widths and a pneumatic microvalve. The device used membrane deformation to control fluid flow and reduce shear stress. Numerical simulations were conducted to assess how device parameters affected pressure drop. A droplet model was applied to evaluate the impact of cell viscosity, size, and channel width on deformation. K562 cells were sorted using filtration channels of 7, 10, 13, and 17 μm widths. The separation efficiency was tested in both media and whole blood. Single cells were trapped and cultured in situ for 4–7 days. Time-lapse imaging captured lineage trees and growth curves to study cell division and size changes.

The filtration channels successfully sorted K562 cells into four size ranges with low driving pressure. The maximum separation efficiency in media was 98.6%, and in whole blood, it was 89.7%. The pneumatic valve effectively controlled fluid flow without causing cell deformation. Numerical simulations confirmed that channel width and cell size influenced pressure drop. The droplet model showed that cell viscosity played a role in deformation during flow. Time-lapse imaging revealed that cells in G1 and G2 phases had distinct sizes. The first cell division time varied among sorted cells. Cell fusion and size variation before and after division were observed. These findings suggest a correlation between cell size and cell cycle phases.

The authors propose that the microfluidic device effectively sorts cells by size and enables in situ clonal expansion. The system’s pneumatic valve minimizes shear stress and maintains cell viability during long-term culture. The filtration channels with varying widths allow for precise sorting of K562 cells. The separation efficiency in whole blood supports potential clinical applications. Time-lapse imaging confirmed that cell size correlates with cell cycle phases. The device’s ability to track lineage trees and growth curves is a key advantage. The findings suggest that this system can be used for detailed single-cell studies. The authors suggest that the device has significant potential in biomedical research and cell therapy.