Challenges in 3D culturing of neutrophils: Assessment of cell viability.

Standard cell culturing on plastic plates (two dimensional (2D) cultures) does not represent the actual microenvironment where cells reside in tissues. The three dimensional (3D) systems, composed of extracellular matrix and/or pure amino acids which form a scaffold for cells, are more accurate in this respect. 3D cultures were primarily developed for cancer cells but there is also a need for their application in studies on inflammatory leukocytes. Herein we describe our approach to study neutrophil-like cells in the 3D system. We describe measures taken to establish a neutrophil-like cell line (nHL-60) and selection of 3D scaffolds (PuraMatrix alone or enriched with collagen type I) for their culturing. We focus on challenges in measurement of neutrophil viability in 3D cultures and based on our data we suggest application of resazurin, rather than tetrazolium-based dyes or trypan blue exclusion, for evaluation of neutrophil viability.