Label-free Detection and Characterization of Metabolism in Fresh and Cryopreserved Macrophages

Cryopreservation is a widely used technique to preserve biological samples for extended periods of time at low temperatures. Even though it is known to have significant effects on cell viability, its effect on their metabolism remains unexplored. Studying how cryopreservation influences the metabolism of cells is important to guarantee the reliability of samples transported between sites for analysis. Optical metabolic imaging allows for the study of cellular metabolism in a label-free manner by using the autofluorescence properties of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), two metabolic coenzymes. The goal of this research is to study the metabolic changes in macrophages after cryopreservation and compare these results with freshly isolated macrophage samples to evaluate if the metabolic data is retained after cryopreservation. The metabolism of macrophages was analyzed with fluorescence lifetime imaging microscopy (FLIM) using a multiphoton microscope. Monocytes were isolated from human whole blood and separated into two groups. In Group 1, freshly isolated monocytes were differentiated into macrophages using macrophage-colony stimulating factor (M-CSF) over 8 days. In Group 2, isolated monocytes were cryopreserved after separation from whole blood and then thawed and stimulated with M-CSF for 8 days. Single-cell analysis showed there are significant changes in FLIM parameters between the groups suggesting that the metabolic data of macrophages is altered after a cryopreservation and cell thawing cycle. Our research bridges the current gap by studying the metabolic changes of cells after cryopreservation using a non-invasive and label-free imaging technique.