Rotating Intern Ontario Veterinary College, University of Guelph Guelph, ON, Canada
Abstract: Background Mesenchymal stem cells (MSCs) exhibit anti-inflammatory properties in cats. A 3D culture technique has demonstrated enhanced effects compared to conventional 2D cultures. Hypothesis/Objectives This study aimed to develop a 3D culture method for feline adipose tissue-derived MSCs (fAT-MSCs) and to compare their anti-inflammatory potential with that of 2D-cultured MSCs. Animals Feline adipose tissue was collected from healthy spayed and neutered donors at Seoul National University Veterinary Medical Teaching Hospital under an approved protocol. Methods fAT-MSC spheroids were generated using an ultra-low-attachment plates. Confocal imaging assessed their structure and hypoxia. Stemness-related proteins (SOX2, OCT4) were analyzed via immunohistochemistry and western blotting. RT-PCR evaluated inflammation-related factors. Conditioned media (CM) from 2D- and 3D-cultured fAT-MSCs were tested on LPS-stimulated Raw264.7 macrophages. Inflammatory responses were assessed by flow cytometry and RT-PCR. Results Hypoxic conditions in 3D cultures significantly enhanced the expression of stemness-related proteins (p < 0.01). Additionally, inflammation-related factors, including TGFβ, TNFα, COX-2, and IL-10 (p < 0.05), were significantly upregulated in 3D-cultured fAT-MSC spheroids. CM from 3D cultures significantly reduced M1 polarization in Raw264.7 cells and decreased inflammatory cytokine expression compared to LPS treatment alone. Conclusions and Clinical Importance This study presents a novel 3D culture method for fAT-MSCs, demonstrating their superior anti-inflammatory effects over 2D cultures. These findings suggest that 3D-cultured fAT-MSCs may serve as a more effective therapeutic approach for inflammatory diseases in cats.
