ISSN: 2577-4360
Authors: Chauhan A and Kumar Gautam P*
Introduction: There are several ways to isolate MSCs, such as using immunomagnetic kits. However, these approaches are costly and not always suitable for large-scale or frequent animal studies. This study aimed to provide a cost-effective, reproducible protocol, purification, and functional characterization of BM-MSCsresearch. Materials and Methods: Swiss albino mice (8-12 weeks, 20-25 g) were housed in pathogen-free conditions in accordance with IAEC rules. We used morphological evaluation, fluorescence microscopy, and flow cytometry to look at BM-MSCs and find MSC markers (CD44, CD90, and CD105) and negative markers (CD45 and CD31). Quantitative real-time PCR was used to look at the gene expression of epithelial-mesenchymal transition (EMT) and tumor-associated markers (N-cadherin, Nectin, Notch). Results: BM-MSCs were successfully isolated, initially exhibiting a rounded morphology that progressively transitioned into a spindle-shaped fibroblastic configuration by days 3 to 5, reaching 60 to 70% confluence by day 7. ICC of P-3 BM-MSCs demonstrated strong expression of mesenchymal markers CD44 and CD90. Flow cytometry analysis revealed a progressive enrichment of MSCs over passages, with CD44⁺/CD105⁺ cells increasing from 0.059% at P-1 to 71.9% at P-2 and ultimately reaching 92.0% at P-3, while remaining negative for CD31 and CD45. Conclusion: This study establishes a comprehensive, reproducible protocol for isolating and characterizing MSCs and generating tumor-conditioned MSCs and validating phenotypic. The improved method provides a substantial foundation for investigating MSC-tumor interactions and developing MSC-based therapeutic strategies.
Keywords: Bone Marrow Mesenchymal Stem Cells; Tumor-Conditioned Mscs; EMT Markers; Flow Cytometry; Immunocytochemistry; Breast Cancer