Single cell barcode microchip (SCBC) has become an informative tool for comprehensive monitoring of the immune effector functions of single cells and has great value for quantifying cellular heterogeneity at the functional level in the complex biological systems such as the immune system or tumor microenvironment [1-4]. Despite its rapid progresses, efforts to control the three dimensional microenvironment for single cell measurements have been lacking . We developed a simple method to incorporate three dimensional scaffolds with PDMS stencil into a multiplexed single cell secretomic analysis platforms to mimic the extracellular physical matrix and mechanical support for single cells . Applying this platform to brain tumor cell line U87 to investigate single cell protein secretion behavior on different substrates, we revealed that single cell protein secretions were regulated differently in 3D microenvironments. The finding was further verified with intracellular cytokine staining, highlighting the significance of 3D single cell microenvironments. This new single cell biomimetic platform can be easily adaptable to other single cell assays and may become a broadly applicable three dimensional single cell analysis system to study the effect of microenvironment conditions on cellular functional heterogeneity in vitro.
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