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1. Scaffold-free development of multicellular tumor spheroids with spatial characterization of structure and metabolic radial profiles

   https://doi.org/10.1007/s44164-024-00074-3  

   Bess, Shelby N; Smart, Gaven K; Igoe, Matthew J; Muldoon, Timothy J (June 2024, In vitro models)

   Abstract PurposeIn vitroassays are essential for studying cellular biology, but traditional monolayer cultures fail to replicate the complex three-dimensional (3D) interactions of cells in living organisms. 3D culture systems offer a more accurate reflection of the cellular microenvironment. However, 3D cultures require robust and unique methods of characterization. MethodsThe goal of this study was to create a 3D spheroid model using cancer cells and macrophages, and to demonstrate a custom image analysis program to assess structural and metabolic changes across spheroid microregions. ResultsStructural characterization shows that cells at the necrotic core show high normalized fluorescence intensities of CD206 (M2 macrophages), cellular apoptosis (cleaved caspase-3, CC3), and hypoxia (HIF-1α and HIF-2α) compared to the proliferative edge, which shows high normalized fluorescence intensities of CD80 (M1 macrophages) and cellular proliferation (Ki67). Metabolic characterization was performed using multiphoton microscopy and fluorescence lifetime imaging (FLIM). Results show that the mean NADH lifetime at the necrotic core (1.011 ± 0.086 ns) was lower than that at the proliferative edge (1.105 ± 0.077 ns). The opposite trend is shown in the A1/A2 ratio (necrotic core: 4.864 ± 0.753; proliferative edge: 4.250 ± 0.432). ConclusionOverall, the results of this study show that 3D multicellular spheroid models can provide a reliable solution for studying tumor biology, allowing for the evaluation of discrete changes across all spheroid microregions. 

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2. Polarized macrophages modulate cardiac structure and contractility under hypoxia in novel immuno-heart on a chip

   https://doi.org/10.1063/5.0253888  

   Schmidt, Andrew A; David, Li-Mor; Qayyum, Nida T; Tran, Khanh; Van, Cassandra; Hetta, Ali_H_S_H A; Shrestha, Ronit L; Varatip, Ashley O; Butenko, Sergei; Enriquez-Ochoa, Daniela; et al (June 2025, APL Bioengineering)

   Cardiac adaptation to hypoxic injury is regulated by dynamic interactions between cardiomyocytes and macrophages, yet the impacts of immune phenotypes on cardiac structure and contractility remain poorly understood. To address this, we developed the immuno-heart on a chip, a novel in vitro platform to investigate cardiomyocyte–macrophage interactions under normoxic and hypoxic conditions. By integrating neonatal rat ventricular myocytes (NRVMs) and bone marrow-derived macrophages—polarized to pro-inflammatory (M1) or pro-healing (M2/M2*) phenotypes—we elucidated the dual protective and detrimental roles macrophages play in modulating cardiomyocyte cytoskeletal architecture and contractility. Pro-inflammatory stimulation reduced cardiomyocyte structural metrics (z-line length, fraction, and integrity) in normoxic co-cultures. Under hypoxia, M1-stimulated NRVM monocultures exhibited declines in cytoskeletal organization—quantified by actin and z-line orientational order parameters. Relative to monocultures, M1-stimulated co-cultures attenuated hypoxia-induced active stress declines but produced weaker normoxic stresses. In contrast, pro-healing stimulation improved normoxic z-line metrics and preserved post-hypoxia cytoskeletal organization but reduced normoxic contractility. Notably, M2-stimulated macrophages restored normoxic contractility and preserved post-hypoxia systolic stress, albeit with increased diastolic stress. RNAseq analysis of M2-stimulated co-cultures identified upregulated structural and immune pathways driving these hypoxia-induced changes. Cytokine profiles revealed stimulation-specific and density-dependent tumor necrosis factor-alpha and interleukin-10 secretion patterns. Together, these findings quantitatively link clinically relevant macrophage phenotypes and cytokines to distinct changes in cardiac structure and contractility, offering mechanistic insights into immune modulation of hypoxia-induced dysfunction. Moreover, the immuno-heart on a chip represents an innovative framework to guide the development of future therapies that integrate immune and cardiac targets to enhance patient outcomes. 

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3. Deriving a Boolean dynamics to reveal macrophage activation with in vitro temporal cytokine expression profiles

   https://doi.org/10.1186/s12859-019-3304-5  

   Ramirez, Ricardo; Herrera, Allen Michael; Ramirez, Joshua; Qian, Chunjiang; Melton, David W.; Shireman, Paula K.; Jin, Yu-Fang (December 2019, BMC Bioinformatics)

   null (Ed.)

   Abstract Background Macrophages show versatile functions in innate immunity, infectious diseases, and progression of cancers and cardiovascular diseases. These versatile functions of macrophages are conducted by different macrophage phenotypes classified as classically activated macrophages and alternatively activated macrophages due to different stimuli in the complex in vivo cytokine environment. Dissecting the regulation of macrophage activations will have a significant impact on disease progression and therapeutic strategy. Mathematical modeling of macrophage activation can improve the understanding of this biological process through quantitative analysis and provide guidance to facilitate future experimental design. However, few results have been reported for a complete model of macrophage activation patterns. Results We globally searched and reviewed literature for macrophage activation from PubMed databases and screened the published experimental results. Temporal in vitro macrophage cytokine expression profiles from published results were selected to establish Boolean network models for macrophage activation patterns in response to three different stimuli. A combination of modeling methods including clustering, binarization, linear programming (LP), Boolean function determination, and semi-tensor product was applied to establish Boolean networks to quantify three macrophage activation patterns. The structure of the networks was confirmed based on protein-protein-interaction databases, pathway databases, and published experimental results. Computational predictions of the network evolution were compared against real experimental results to validate the effectiveness of the Boolean network models. Conclusion Three macrophage activation core evolution maps were established based on the Boolean networks using Matlab. Cytokine signatures of macrophage activation patterns were identified, providing a possible determination of macrophage activations using extracellular cytokine measurements. 

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4. Immunomodulation Through Fibroblast-Derived Extracellular Vesicles (EVs) Within 3D Polycaprolactone–Collagen Matrix

   https://doi.org/10.3390/biomimetics10080484  

   Tasnim, Afsara; Jacho, Diego; Rabino, Agustin; Benalcazar, Jose; Garcia-Mata, Rafael; Lapitsky, Yakov; Yildirim-Ayan, Eda (August 2025, Biomimetics)

   Extracellular vesicles (EVs) have emerged as promising acellular tools for modulating immune responses for tissue engineering applications. This study explores the potential of human fibroblast-derived EVs delivered within a three-dimensional (3D) injectable scaffold composed of polycaprolactone (PCL) nanofibers and collagen (PNCOL) to reprogram macrophage behavior and support scaffold integrity under inflammatory conditions. EVs were successfully isolated from human fibroblasts using ultracentrifugation and characterized for purity, size distribution and surface markers (CD63 and CD9). Macrophage-laden PNCOL scaffolds were prepared under three conditions: macrophage-only (MP), fibroblast co-encapsulated (F-MP), and EV-encapsulated (EV-MP) groups. Structural integrity was assessed via scanning electron microscopy and Masson’s trichrome staining, while immunomodulatory effects were evaluated through metabolic assays, gene expression profiling, and immunohistochemistry for macrophage polarization markers (CD80, CD206). When co-encapsulated with pro-inflammatory (M1) macrophages in PNCOL scaffolds, fibroblast-derived EVs preserved scaffold structure and significantly enhanced macrophage metabolic activity compared to the control (MP) and other experimental group (F-MP). The gene expression and immunohistochemistry data demonstrated substantial upregulation of anti-inflammatory markers (TGF-β, CD163, and CCL18) and surface protein CD206, indicating a phenotypic shift toward M2-like macrophages for EV-encapsulated scaffolds relative to the other groups. The findings of this study demonstrate that fibroblast-derived EVs integrated into injectable PCL–collagen scaffolds offer a viable, cell-free approach to modulate inflammation, preserve scaffold structure, and support regenerative healing. This strategy holds significant promise for advancing immuno-instructive platforms in regenerative medicine, particularly in settings where conventional cell therapies face limitations in survival, cost, or safety. 

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5. Light and metabolism: label-free optical imaging of metabolic activities in biological systems [Invited]

   https://doi.org/10.1364/BOE.572304  

   Menozzi, Luca; Li, Zhi; Choi, Seongwook; Vu, Tri; Shi, Lingyan; Yao, Junjie (January 2025, Biomedical Optics Express)

   Metabolic imaging is critical for understanding cellular functions beyond morphology, offering significant insights into various biological processes and disease states. Label-free optical imaging techniques stand out by providing high-resolution, molecularly specific, and/or non-invasive assessments of metabolic activity without relying on exogenous contrast agents. This review discusses the key photon-tissue interactions—absorption, emission, and scattering—that underpin label-free optical imaging modalities for interrogating tissue’s metabolic activities at various scales. Specifically, photoacoustic imaging (PAI) leverages absorption-based contrasts such as hemoglobin oxygenation and glucose concentrations to quantify metabolic dynamics. Emission-based techniques, including two-photon fluorescence (TPF) and fluorescence lifetime imaging microscopy (FLIM), exploit intrinsic fluorophores like nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) to assess cellular energy metabolism. Interferometric methods, particularly optical coherence tomography (OCT), provide insights into tissue morphological changes. Second harmonic generation (SHG) detects extracellular matrix components such as the collagen network. Molecular vibrational imaging methods, such as stimulated Raman scattering (SRS) microscopy, visualizes spatial heterogeneity of molecular compositions. Recent clinical translations of these methods highlight their growing roles in oncology, neurology, and dermatology, underscoring their potential in early disease diagnosis and monitoring therapeutic responses. Despite challenges such as depth limitations, advancements like wavefront engineering and optical clearing techniques promise to enhance imaging penetration and clinical applicability, paving the way for broader adoption of label-free optical metabolic imaging in both research and clinical settings. 

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