Avicenna Veterinary Research

Avicenna Veterinary Research

Optimization of a Culture Protocol for Murine Bone Marrow-Derived Mesenchymal Stromal Cells

Document Type : Original Article

Authors
Mohammad Hossein Pouria Far
Sahar Hamoonnavard
Hossein Rezvan
Department of Pathobiology, Faculty of Veterinary Medicine, Bu-Ali Sina University, Hamedan, Iran
10.22084/avr.2026.32294.1032
Abstract
Background: Mesenchymal stem cells (MSCs) have attracted extensive research due to their high proliferative capacity, multipotent differentiation, and essential role in tissue regeneration. Despite their application in clinical research, there are limitations in the culture and maintenance of these cells. This study aimed to optimize a cost-effective and efficient protocol for the culture and expansion of M-MSCs. Methods: MSCs were isolated from BALB/c mouse femoral bone marrow (6{8 weeks old) and cultured in DMEM-Low Glucose with 20%-5% fetal bovine serum (FBS), penicillin/streptomycin (1%), and gentamicin (0.5%). At passage 3, cells exhibited broblastic morphology and were immunopheno-typed for MSCs (CD73-PE, CD105-PerCP-Cy5.5) and hematopoietic markers (CD34-PE, CD45-FITC). The growth rate, post-freezing cell viability, and population doubling time were evaluated. Results: Fibroblastic morphology was observed at 48 hours post-primary culture, with cells forming a scaffold and reaching 80% con uency by day 5. Under 5% FBS conditions, the time to reach 90% con uency decreased to<5 days by passages 2 and 3. Flow cytometry con rmed a typical MSCs pro le: high CD73 (99.9%) and CD105 (98.5%) and low CD34 (5.99%) and CD45 (2.86%) expression. Results show a positive correlation between increasing passage number and enhanced MSCs growth rate, viability and functional characteristics (p0.05). Conclusion: Optimization of M-MSCs culture through a FBS reduction (20% to 5%) is shown to preserve key cellular properties: broblastic morphology, proliferation rate, and immunophenotypic markers. Our protocol reduces microbial contamination associated with animal serum and provides an economical, and viable strategy for large scale M-MSCs production in regenerative medicine.
Keywords
Murine mesenchymal stem cells
fetal bovine serum
culture optimization
immunophenotype
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Avicenna Veterinary Research
Volume 2, Issue 2
Spring 2026
Pages 9-16