Label‐Free Cell Viability Assay and Enrichment of Cryopreserved Cells Using Microfluidic Cytometry and On‐Demand Sorting

Abstract Cryopreservation is essential for the long‐term storage of primary cell samples and enables accessing primary cells anytime via a simple thawing process. However, low viability and cell fragmentation are the major issues for cryopreserved cell samples due to the toxicity of cryoprotective agents and crystallization of intracellular contents during cooling at low temperature. Human peripheral blood mononuclear cells (PBMCs) in high viability are critical for pathophysiological and therapeutic research of infectious diseases, especially for the current worldwide pandemic of coronavirus disease 2019. Conventional viability enrichment techniques for primary PBMC samples provoke either cryoinjury caused by cell sorting mechanism or substantial cell loss due to multiple sample preparation steps. Here, a label‐free viability assessment and on‐demand enrichment system for cryopreserved primary human PBMCs with validation by fluorescent label‐based flow cytometry, is demonstrated. It can provide consistent viability assays by discriminating viable PBMCs from apoptotic, DMSO toxicity‐induced dead cells and cell debris, and deliver over 90% viability of sorted samples in one step after thawing. This integrated cell viability assay and on‐demand enrichment of viable cells has broad applications in diverse cellular biology research and cell‐related biomedical diagnostics.