Short-Term Cryopreservation Preserved the Function of MSCs from Bone Marrow Aspirate Concentrate
Abstract
Highlights
- MSC proliferation and multilineage differentiation were preserved after freezing BMAC at −80 °C for 4 weeks.
- Both fresh and frozen BMAC equally improved histological cartilage scores compared with PBS control in an OA rat model.
- Frozen BMAC retains functional equivalence to fresh BMAC for cartilage repair.
- A single bone marrow harvest with storage for multiple injections may reduce patient burden and expand clinical utility.
Abstract
1. Introduction
2. Materials and Methods
2.1. Patient Recruitment and BMAC Collection
2.2. Preparation of Frozen BMAC and Plasma
2.3. Mononuclear Cell Isolation from Fresh and Frozen BMAC and Platelets Measurement
2.4. Colony Forming Units-Fibroblast (CFU-f) Assay
2.5. Multiplex Analysis to Detect Soluble Factors in the Plasma from BMAC
2.6. In Vitro Adipogenic Differentiation Assay and Oil Red O Staining
2.7. In Vitro Osteogenic Differentiation Assay and Alizarin Red Staining
2.8. In Vitro 3D Pellet Culture for Chondrogenic Differentiation
2.9. Immunohistochemistry
2.10. q-PCR Analysis
2.11. Surgical Induction of Osteoarthritis and Human BMAC Treatment
2.12. Micro-CT Scanning
2.13. Histology
2.14. Statistical Analysis
3. Results
3.1. Effect of Freezing on Platelets in the BMAC
3.2. Effect of Freezing on CFU-f Formation
3.3. Effect of Freezing on Adipogenic and Osteogenic Differentiation of MSCs and Markers of MSC
3.4. Effects of Freezing on Chondrogenic Differentiation of MSCs
3.5. Effect of Freezing on Soluble Factors in the Plasma from BMAC
3.6. No Significant Difference Between Fresh and Frozen in the AC Regenerative Potential of BMAC
3.7. Neither Fresh nor Frozen BMAC Treatment Significantly Affected Epiphysis and Subchondral Bone
3.8. Both Fresh and Frozen BMAC Treatment Improved the Histology Score of Tibial Plateau Cartilage
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene Name | Primer Sequence (5′-3′) | Product Size (bp) |
---|---|---|
Gapdh | Forward: GTATCGGACGCCTGGTTACC Reverse: ACCAGCTTCCCATTCTCAGC | 166 |
CD90 | Forward: CAGCAGTTCACCCATCCAGT Reverse: GATGCCCTCACACTTGACCA | 271 |
Sox2 | Forward: GCTACAGCATGATGCAGGACCA Reverse: TCTGCGAGCTGGTCATGGAGTT | 134 |
OCT4 | Forward: GCAAAGCAGAAACCCTCGTG Reverse: AACCACACTCGGACCACATC | 172 |
MMP9 | Forward: ACAGCGAGACACTAAAGGCC Reverse: GGCAAGTCTTCGGTGTAGCT | 139 |
MMP13 | Forward: TCCATCCCGAGACCTCATGT Reverse: CACACGTGGTTCCCTGAGAA | 189 |
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Singer, J.; Nishimura, H.; Xiao, Z.; Gao, X.; Knezic, N.; Chubb, L.; Layne, J.E.; Guo, P.; Lu, A.; Huard, J. Short-Term Cryopreservation Preserved the Function of MSCs from Bone Marrow Aspirate Concentrate. Cells 2025, 14, 1569. https://doi.org/10.3390/cells14191569
Singer J, Nishimura H, Xiao Z, Gao X, Knezic N, Chubb L, Layne JE, Guo P, Lu A, Huard J. Short-Term Cryopreservation Preserved the Function of MSCs from Bone Marrow Aspirate Concentrate. Cells. 2025; 14(19):1569. https://doi.org/10.3390/cells14191569
Chicago/Turabian StyleSinger, Jacob, Haruki Nishimura, Zuokui Xiao, Xueqin Gao, Noah Knezic, Laura Chubb, Jonathan E. Layne, Ping Guo, Aiping Lu, and Johnny Huard. 2025. "Short-Term Cryopreservation Preserved the Function of MSCs from Bone Marrow Aspirate Concentrate" Cells 14, no. 19: 1569. https://doi.org/10.3390/cells14191569
APA StyleSinger, J., Nishimura, H., Xiao, Z., Gao, X., Knezic, N., Chubb, L., Layne, J. E., Guo, P., Lu, A., & Huard, J. (2025). Short-Term Cryopreservation Preserved the Function of MSCs from Bone Marrow Aspirate Concentrate. Cells, 14(19), 1569. https://doi.org/10.3390/cells14191569