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Abstract BackgroundMacrophages are one of the most prevalent subsets of immune cells within the tumor microenvironment and perform a range of functions depending on the cytokines and chemokines released by surrounding cells and tissues. Recent research has revealed that macrophages can exhibit a spectrum of phenotypes, making them highly plastic due to their ability to alter their physiology in response to environmental cues. Recent advances in examining heterogeneous macrophage populations include optical metabolic imaging, such as fluorescence lifetime imaging (FLIM), and multiphoton microscopy. However, the method of detection for these systems is reliant upon the coenzymes NAD(P)H and FAD, which can be affected by factors other than cytoplasmic metabolic changes. In this study, we seek to validate these optical measures of metabolism by comparing optical results to more standard methods of evaluating cellular metabolism, such as extracellular flux assays and the presence of metabolic intermediates. MethodsHere, we used autofluorescence imaging of endogenous metabolic co-factors via multiphoton microscopy and FLIM in conjunction with oxygen consumption rate and extracellular acidification rate through Seahorse extracellular flux assays to detect changes in cellular metabolism in quiescent and classically activated macrophages in response to cytokine stimulation. ResultsBased on our Seahorse XFP flux analysis, M0 and M1 macrophages exhibit comparable trends in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Autofluorescence imaging of M0 and M1 macrophages was not only able to show acute changes in the optical redox ratio from pre-differentiation (0 hours) to 72 hours post-cytokine differentiation (M0: 0.320 to 0.258 and M1: 0.316 to 0.386), mean NADH lifetime (M0: 1.272 ns to 1.379 ns and M1: 1.265 ns to 1.206 ns), and A1/A2 ratio (M0: 3.452 to ~ 4 and M1: 3.537 to 4.529) but could also detect heterogeneity within each macrophage population. ConclusionsOverall, the findings of this study suggest that autofluorescence metabolic imaging could be a reliable technique for longitudinal tracking of immune cell metabolism during activation post-cytokine stimulation.
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This content will become publicly available on April 9, 2026
Microfluidic chip-based co-culture system for modeling human joint inflammation in osteoarthritis research
Here we present a microfluidic model that allows for co-culture of human osteoblasts, chondrocytes, fibroblasts, and macrophages of both quiescent (M0) and pro-inflammatory (M1) phenotypes, maintaining initial viability of each cell type at 24 h of co-culture. We established healthy (M0-based) and diseased (M1-based) joint models within this system. An established disease model based on supplementation of IFN-γ and lipopolysaccharide in cell culture media was used to induce an M1 phenotype in macrophages to recapitulate inflammatory conditions found in Osteoarthritis. Cell viability was assessed using NucBlue™ Live and NucGreen™ Dead fluorescent stains, with mean viability of 83.9% ± 14% and 83.3% ± 12% for healthy and diseased models, respectively, compared with 93.3% ± 4% for cell in standard monoculture conditions. Cytotoxicity was assessed via a lactate dehydrogenase (LDH) assay and showed no measurable increase in lactate dehydrogenase release into the culture medium under co-culture conditions, indicating that neither model promotes a loss of cell membrane integrity due to cytotoxic effects. Cellular metabolic activity was assessed using a PrestoBlue™ assay and indicated increased cellular metabolic activity in co-culture, with levels 5.9 ± 3.2 times mean monolayer cell metabolic activity levels in the healthy joint model and 5.3 ± 3.4 times mean monolayer levels in the diseased model. Overall, these findings indicate that the multi-tissue nature ofin vivohuman joint conditions can be recapitulated by our microfluidic co-culture system at 24 h and thus this model serves as a promising tool for studying the pathophysiology of rheumatic diseases and testing potential therapeutics.
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- Award ID(s):
- 2517512
- PAR ID:
- 10623864
- Publisher / Repository:
- Frontiers
- Date Published:
- Journal Name:
- Frontiers in Pharmacology
- Volume:
- 16
- ISSN:
- 1663-9812
- Page Range / eLocation ID:
- 1579228
- Subject(s) / Keyword(s):
- osteoarthritis microfluidic co-culture human joint model in vitro disease modeling
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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