In vitro culture of single cells facilitates biological studies by de-convoluting complications from cell population heterogeneity.However,there is still a lack of simple yet high-throughput methods to for single cell culture and analysis.Moreover,most of the single-cell culture and analysis devices are based on 2D cell culture conditions,even though 3D cell culture methods have been demonstrated to better mimic the real cell microenvironment in vivo.To solve these problems.We propose the development and application of a multifunctional droplet microchip with a u-Fluidic trap chip for high single cell capture efficiency(~90%)in micro-chamber which allowed for cell spreading,proliferation and differentiation.The method capitalizes on the unique thermo-responsive sol-gel switching property of agarose for cell suspension,single cell capture,and long term 3D culture.This novel device architecture allows the size of the "culture" micro-chamber to be flexibly adjusted without affecting the single-cell loading efficiency.We have demonstrated the single-cell culture and single-cell colony formation with 293T cells.The agarose droplet array could be further applied for study of cellular proliferation heterogeneity and drug cytotoxicity at the single-cell level.