High‐Throughput Single‐Cell Analysis of Local Nascent Protein Deposition in 3D Microenvironments via Extracellular Protein Identification Cytometry (EPIC)

Extracellular matrix (ECM) guides cell behavior and tissue fate. Cell populations are notoriously heterogeneous leading to large variations in cell behavior at the single-cell level. Although insights into population heterogeneity are valuable for fundamental biology, regenerative medicine, and drug testing, current ECM analysis techniques only provide either averaged population-level data or single-cell data from a limited number of cells. Here, extracellular protein identification cytometry (EPIC) is presented as a novel platform technology that enables high-throughput measurements of local nascent protein deposition at single-cell level. Specifically, human primary chondrocytes are microfluidically encapsulated in enzymatically crosslinked microgels of 16 picoliter at kHz rates, forming large libraries of discrete 3D single-cell microniches in which ECM can be deposited. ECM proteins are labeled using fluorescence immunostaining to allow for nondestructive analysis via flow cytometry. This approach reveals population heterogeneity in matrix deposition at unprecedented throughput, allowing for the identification and fluorescent activated cell sorting-mediated isolation of cellular subpopulations. Additionally, it is demonstrated that inclusion of a second cell into microgels allows for studying the effect of cell-cell contact on matrix deposition. In summary, EPIC enables high-throughput single-cell analysis of nascent proteins in 3D microenvironments, which is anticipated to advance fundamental knowledge and tissue engineering applications.