bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.01.13.632815

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

Canales, D. D. H.·Hu, L.·Walsh, A.
View original source ↗Export metadata ↓

Abstract

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.

Explore related subjects

bioengineeringPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Canales, D. D. H., Hu, L., Walsh, A.. 2025-01-17. Label-free Detection and Characterization of Metabolism in Fresh and Cryopreserved Macrophages. https://doi.org/10.1101/2025.01.13.632815

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Surfactant-Assisted Colorimetric Signal Enhancement in Paper-Based Glucose Sensing

Paper-based colorimetric sensors offer a low-cost and accessible platform for point-of-care (POC) analysis, but enzyme activity loss during coating and drying can weaken analytical signals and require high enzyme loadings or complex immobilization procedures. Although surfactants are widely used to improve wettability in paper-based assays, their potential contribution to colorimetric performance beyond these effects remains unclear. Here, we investigated surfactant-assisted colorimetric signal enhancement in a glucose assay implemented on a 96-puddle paper plate (96-PPP) and identified Tween 20 as the most effective surfactant. Its effect on detection performance became more pronounced as glucose oxidase (GOx) loading decreased; at 0.1 mg/mL GOx, Tween 20 lowered the limit of detection (LoD) from 0.113 to 0.034 mg/mL (approximately 3.3-fold) over a working range of 0-5 mg/mL, despite no statistically significant change in the measured contact angle at this loading. Tween 20 had no appreciable effect on the reaction in solution but preserved 95% of the apparent reaction rate constant after drying, compared with 11% without it, and atomic force microscopy (AFM) revealed a more dispersed dried enzyme morphology on mica. Tween 20-containing sensors also showed slower signal decay during repeated wetting-drying cycles and thermal stress, retained 77% (vs 26%) of the response at 400 mM NaCl, and exhibited within-PPP and between-batch coefficients of variation (CVs) below 10% (vs 12.3-19.5%), while maintaining glucose selectivity over potentially interfering molecules. These results indicate that Tween 20 enhances paper-based glucose sensing beyond wettability, in part by retaining enzyme cascade activity during drying, although the contributions of the individual enzymes and the underlying mechanism remain to be established.

bioengineering↗
bioRxiv2026

Engineering CAR-T cells to remodel the mucin-rich cancer cell glycocalyx

The dense glycocalyx of cancer cells can restrict immune-cell access to surface antigens and limit CAR-T cell activity. Here, we show that mucin density and epitope position determine how glycocalyx remodeling affects CAR-T cell recognition and killing. We identify KLK5 as a human protease that cleaves tumor-associated mucins, increases access to membrane-proximal antigens, and enhances CAR-T cell function. We then engineer CAR-T cells to display or secrete KLK5, enabling remodeling of the tumor glycocalyx during antigen recognition. KLK5-engineered CAR-T cells improved tumor control across multiple xenograft models, and KLK5-secreting MUC17 CAR-T cells produced the strongest in vivo benefit, prolonging survival compared with conventional MUC17 CAR-T cells. These findings show that CAR-T cells can be engineered to breach the mucin-rich glycocalyx while preserving accessible target epitopes.

bioengineering↗
bioRxiv2026

Wall stiffening is a primary contributor to motility loss in Crohn's disease: an electromechanical modeling study

Fibrotic strictures are among the most disabling complications of Crohn's disease, permanently narrowing the bowel and impairing motility, yet no approved therapy reverses them. Chronic inflammation alters pacemaker-network coupling, smooth-muscle excitability, and calcium-dependent contractility, while fibrosis thickens the bowel wall, narrows the lumen, and changes tissue mechanics. The relative contributions of these coupled electrical, contractile, and structural alterations to motility loss remain unclear. To address this gap, we develop an integrated electromechanical finite-element framework for fibrostenosing Crohn's disease that couples a fibrosis-driven growth model with a FitzHugh-Nagumo electromechanical model. A full-factorial 25 design of experiments is used to quantify the relative effects of electrical diffusivity, excitation threshold, peak active stress, wall stiffness, and hypertrophic remodeling on cyclic lumen-volume deformation. Motility is quantified by the standard deviation of lumen volume over one contraction cycle. Within the parameter ranges examined, increased wall stiffness emerged as the dominant contributor to motility loss, followed by impaired smooth-muscle contractility. Changes in excitation threshold, hypertrophic remodeling, and electrical diffusivity produced substantially smaller effects. Pairwise interactions were small relative to the dominant main effects, indicating that the mechanisms contributed largely through their individual effects. Our findings suggest that limiting wall stiffening while preserving smooth-muscle contractile function may provide a therapeutic strategy for maintaining intestinal motility in fibrostenosing Crohn's disease.

bioengineering↗