The method to dynamically screen and print single cells using microfluidics with pneumatic microvalves.

Printing single cells into individual chambers is of critical importance for single-cell analysis using traditional equipment, for instance, single-cell clonal expansion or sequencing. The size of cells can usually be a reflection of their types, functions, and even cell cycle phases. Therefore, printing individual cells within the desired size range is of essential application potential in single-cell analysis. This paper presents a method for the development of a microfluidic chip integrating pneumatic microvalves to print single cells with appropriate size into standard well plates. The reported method provided essential guidelines for the fabrication of multi-layer microfluidic chips, control of the membrane deflection to screen cell size, and printing of single cells. In brief, this paper reports:•the manufacturing of the chip using standard soft lithography;•the protocol to dynamically screen both the lower and the upper size limit of cells passing through the valves by deflection of the valve membrane;•the screening and dispensing of suspended human umbilical vein endothelial cells (HUVECs) into 384-well plates with high viability.