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We provide a detailed protocol for a three-dimensional long-term live imaging of cellular spheroids with light sheet-based fluorescence microscopy. The protocol allows the recording of all phases of spheroid formation in three dimensions, including cell proliferation, aggregation, and compaction. We employ the human hepatic cell line HepaRG transfected with the fusion protein H2B-GFP, i.e., a fluorescing histone. The protocol allows monitoring the effect of drugs or toxicants.
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http://dx.doi.org/10.1007/978-1-4939-2080-8_3 | DOI Listing |
Cell Mol Immunol
September 2025
Department of Gastroenterology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, China.
Gut-derived metabolites are essential for liver fibrogenesis. The aim of this study was to determine the alteration of indole-3-propionic acid (IPA), a crucial tryptophan metabolite, in liver fibrosis and delineate the roles of enterogenic IPA in fibrogenesis. In the present study, metabolomics assays focused on tryptophan metabolism were applied to explore the decreased levels of IPA in the feces and serum of cirrhotic patients, as well as in the feces and portal vein serum of fibrotic mice.
View Article and Find Full Text PDFBiomater Sci
September 2025
Biotechnology Science and Engineering Program, University of Alabama in Huntsville, Huntsville, AL 35899, USA.
B cells are critical components of the adaptive immune system that proliferate and differentiate within the secondary lymphoid organs upon recognition of antigens and engagement of T cells. Traditional two-dimensional (2D) cell cultures fall short of replicating the intricate structures and dynamic evolution of three-dimensional (3D) environments found in lymphoid organs, prompting the development of more physiologically pertinent models. Our approach employs -hexanoyl glycol chitosan (HGC) coated ultra-low attachment (ULA) lattice plates to cultivate a 3D co-culture of CD40L-expressing MS5 stromal cells and naïve B cells derived from the peripheral blood mononuclear cells (PBMCs) of healthy human donors.
View Article and Find Full Text PDFFront Bioeng Biotechnol
August 2025
Department of Biomedical Engineering, Pennsylvania State University, University Park, PA, United States.
Background: Mesenchymal stem cells (MSCs) hold great promise for treating a variety of human diseases; however, their clinical translation is hindered by challenges in large-scale expansion while preserving therapeutic potency and maintaining small cell size. Conventional 2D culture on rigid substrates induces MSC senescence and enlargement, compromising their function and biodistribution.
Methods: We present an alternating 2D/3D culture strategy that combines adherent monolayer expansion with transient spheroid formation to mitigate these limitations.
Biochem Biophys Rep
December 2025
Research Center for Pharmaceutical Nanotechnology, Biomedicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran.
Breast cancer is the most prevalent cancer among women, posing significant challenges due to its heterogeneity. Recent studies suggest that the ketogenic diet (KD) may enhance chemotherapy efficacy by modulating cancer cell metabolism, particularly through the elevation of ketone bodies like β-hydroxybutyrate (BHB). This study investigates the effects of BHB on breast cancer cells using both 2D and 3D culture models, focusing on its role in developing resistance to fluorouracil (5-FU).
View Article and Find Full Text PDFAn integrated approach is proposed to rapidly evaluate the effects of anticancer treatments in 3D models, combining a droplet-based microfluidic platform for spheroid formation and single-spheroid chemotherapy application, label-free morphological analysis, and machine learning to assess treatment response. Morphological features of spheroids, such as size and color intensity, are extracted and selected using the multivariate information-based inductive causation algorithm, and used to train a neural network for spheroid classification into viability classes, derived from metabolic assays performed within the same platform as a benchmark. The model is tested on Ewing sarcoma cell lines and patient-derived xenograft (PDX) cells, demonstrating robust performance across datasets.
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