A Rapid Human Lung Tissue Dissociation Protocol Maximizing Cell Yield and Minimizing Cellular Stress.

Abstract

The human lung is a complex organ that comprises diverse populations of epithelial, mesenchymal, vascular, and immune cells, which gains even greater complexity during disease states. To effectively study the lung at a single-cell level, a dissociation protocol that achieves the highest yield of viable cells of interest with minimal dissociation-associated protein or transcription changes is key. Here, we detail a rapid collagenase-based dissociation protocol (Col-Short) that provides a high-yield single-cell suspension that is suitable for a variety of downstream applications. Diseased human lung explants were obtained and dissociated through the Col-Short protocol and compared with four other dissociation protocols. Resulting single-cell suspensions were then assessed with flow cytometry, differential staining, and quantitative real-time PCR to identify major hematopoietic and nonhematopoietic cell populations, as well as their activation states. We observed that the Col-Short protocol provides the greatest number of cells per gram of lung tissue, with no reduction in viability when compared with previously described dissociation protocols. Col-Short had no observable surface protein marker cleavage as well as lower expression of protein activation markers and stress-related transcripts compared with four other protocols. The Col-Short dissociation protocol can be used as a rapid strategy to generate single cells for respiratory cell biology research.