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Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation - Poster Session C

Poster F10 - Label-free Detection of Metabolic Perturbations in Immune Cells After Cryopreservation

Friday, October 25, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Understanding the effects of cryopreservation on white blood cell (WBC) metabolism is essential for the reliability of samples transported for research or diagnostic purposes. Optical metabolic imaging utilizes the auto-fluorescence properties of NAD(P)H and FAD to study cellular metabolism using a label-free approach. This research study aims to investigate the metabolic alterations in WBCs post-cryopreservation in comparison to fresh samples and examine if the metabolic status of WBCs is retained. The result of this study will be crucial for understanding the feasibility of samples for subsequent analysis.

Materials and

Methods:  Peripheral blood mononuclear cells (PBMCs) were isolated via density gradient separation from whole blood, and subsequent T-cell and monocyte isolation was achieved using RosetteSep™ Human T Cell Enrichment Cocktail and RosetteSep™ Human Monocyte Enrichment Cocktail, respectively. Cultured cells were imaged using a multiphoton fluorescence lifetime microscope equipped with a 100x (1.46 NA) objective. Each NAD(P)H and FAD fluorescence lifetime image (256 × 256 pixels, 270 × 270 µm) was acquired with a pixel dwell time of 50 µs and 5 frame repeats. Imaging sessions were conducted under controlled conditions maintained at 37 ℃ with 85% humidity and CO2 in a stage incubator. FLIM data was analyzed to compare metabolic profiles between fresh and cryopreserved samples at the single cell-level.

Results, Conclusions, and Discussions: The NAD(P)H lifetime variations in T cells on the second day are consistent between fresh and frozen blood samples. Specifically, T cells exhibit an increase in the fraction of free NAD(P)H (a1) and a decrease in mean NAD(P)H lifetime (tm) on the second day. The NAD(P)H lifetime of T-cells showed smaller variations between fresh and frozen/thawed samples compared to macrophages. However, statistically significant changes in NAD(PH) lifetimes were not detected for either macrophages or T cells, to conclude that there was not a significant change in metabolic activity of the cells. Similarly, there were no significant changes in FAD lifetime for both fresh and frozen/thawed T-cells and macrophages.

In conclusion, the study found no significant changes in the FLIM parameters between fresh and cryopreserved samples of fresh blood isolated macrophages and T-cells. This suggests that the metabolic information of WBCs remains largely unchanged following the freezing and thawing cycle. These findings strengthen our confidence in the reliability and suitability of cryopreserved samples for subsequent analysis.

This study facilitates the application of autofluorescence lifetime imaging techniques in measuring the metabolism of fresh white blood cells or frozen/thawed white blood cells. Metabolism is crucial to immune cell function, and cellular metabolic activity is closely related to immune cell activation, proliferation, and differentiation. In response to stimuli, T cells experience metabolic perturbations, specifically relying on glycolysis upon activation. Similarly, macrophages are known for their metabolic flexibility in different polarized phenotypes, which allows them to aid in an inflammatory response or the repair of tissue. Autofluorescence lifetime imaging of fresh or frozen/thawed immune cells therefore will allow analysis of immune cell metabolism and function to study metabolic diseases and develop prognostic or diagnostic tests.

Acknowledgements (Optional):