Culture and Identification Techniques of Rat Bone Marrow Mesenchymal Stem Cells

Vollständiger Abstract

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Introduction: Although current stem cell research is considerably advanced, systematic studies on rat Bone Marrow Mesenchymal Stem Cells (BMSCs) remain limited. Novice researchers often encounter difficulties in cell extraction, culture, and identification. Methods: Tibias and femurs were obtained from 4-week-old Sprague-Dawley rats, and bone marrow was extracted through centrifugation. Cell morphology was monitored from P0 to P5. Flow cytometry was performed to detect surface antigen (CD29, CD44, CD90, CD11b, CD34, CD45) expression in P2-P4 cells. Based on the flow cytometry results, cells of the optimal passage were selected for trilineage differentiation. results: The specialized medium significantly accelerated cell proliferation, requiring only 7 days from P0 to P3. Cells exhibited typical fibroblast-like morphology, forming spindle-shaped vortex-like arrangements at high density. Flow cytometric analysis showed that P2 cell purity was suboptimal (CD34, CD45, CD11b &amp;amp;amp;gt; 2%; CD29, CD90 &amp;amp;amp;lt; 95&amp;amp;amp;gt; 95%, CD90 ~95%). P4 cells demonstrated excellent purity (CD44 100%, CD90 99%, CD29 96.8%), fully complying with international standards. Regarding differentiation, BMSCs in good growth state showed obvious calcium nodules after 12 days of osteogenic induction, with strong positive Alizarin Red S staining. Numerous lipid droplets formed after 10 days of adipogenic induction, with significant Oil Red O staining results. Cell microspheres were completely suspended after 18 days of chondrogenic induction, and Alcian Blue staining revealed abundant acidic mucopolysaccharides. Results: BMSCs were successfully cultured with appropriate culture medium and exhibited typical fibroblast‑like morphology. CCK-8 assays showed significant differences in proliferation among the XR medium, α‑MEM, and DMEM groups (P < 0.05). P4 cells exhibited excellent phenotypic purity: CD29, CD44, and CD90 were 95.17% ± 0.2, 99.97% ± 0.05, and 98.93% ± 0.16; CD34, CD45, and CD11b were 1.20% ± 0.17, 1.37% ± 0.11, and 0.31% ± 0.15. Regarding differentiation assays, healthy BMSCs formed distinct calcium nodules after 12 days of osteogenic induction (Alizarin Red S‑positive), multiple Oil Red O‑positive lipid droplets after 10 days of adipogenic induction, and Alcian blue‑positive pellets after 18 days of chondrogenic induction. Discussion: High-purity and high-viability BMSCs can be stably obtained from 4-week-old SD rats. P4 cells fully complied with the International Society for Cellular Therapy (ISCT) criteria in terms of immunophenotype (CD29⁺/CD44⁺/CD90⁺, CD11b⁻/CD34⁻/CD45⁻) and trilineage (osteogenic, adipogenic, and chondrogenic) differentiation potential. Trypsin containing EDTA interferes with the detection of surface antigens such as CD29. Conclusion: The phenotypic identification and trilineage differentiation of rat BMSCs with high purity and high viability comply with international standards. The use of trypsin containing EDTA should be avoided, as it may interfere with the detection of cell surface antigens.