A Long-Term Human Adipocyte Organoid Model Reveals Aging-Associated Responses to Intermittent Hypoxia.

Abstract

Obstructive sleep apnea (OSA), characterized by recurrent intermittent hypoxia (IH), is increasingly recognized as a driver of adipose tissue dysfunction, insulin resistance, and accelerated aging. However, current in vitro models inadequately recapitulate the long-term effects of IH on human adipocytes. Here, we developed a robust long-term human adipocyte organoid culture system that models IH-induced adipocyte aging in vitro. Human stromal vascular fraction (SVF) cells isolated from subcutaneous abdominal adipose tissue were embedded in Matrigel and seeded into Biofloat U-bottom 96-well plates. Using a 1:1 Matrigel-cell mixture and optimized seeding volumes (5-20 µL), adipocyte organoids formed within 10-12 days and maintained stable morphology and viability for more than 90 days. Matrigel was essential for structural integrity, whereas gelatin and low-melting agarose failed to support organoid formation. Subcutaneous preadipocyte medium supplemented with 10% FBS supported more robust adipogenic differentiation and long-term maintenance than Advanced/F12K medium. To model OSA-associated hypoxic stress, organoids were exposed to programmable IH. IH suppressed adipogenesis, as evidenced by reduced lipid accumulation, downregulation of adipogenic markers (PPARγ, adiponectin, FABP4), and reduced lipid droplets. Transmission electron microscopy revealed IH-induced ultrastructural abnormalities, including endoplasmic reticulum fragmentation, mitochondrial disruption, nuclear enlargement, and heterochromatin accumulation-features consistent with cellular senescence. IH further upregulated HIF1α, H2AX, repressive histone methylation marks (H3K9me3, H3K79me3, H4K20me3), and extracellular matrix remodeling proteins (fibronectin, LOX), while impairing insulin signaling as demonstrated by reduced PI3K and AKT phosphorylation. Collectively, these findings establish a physiologically relevant human adipocyte organoid platform for investigating IH-induced adipocyte dysfunction and aging.