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Density gradient centrifugation compromises bone marrow mononuclear cell yield
Claudia Pösel1, Karoline Möller, Wenke Fröhlich
1Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany. claudia.poesel@izi.fraunhofer.de
Abstract:
Bone marrow mononuclear cells (BMNCs) are widely used in regenerative medicine, but recent data suggests that the isolation of BMNCs by commonly used Ficoll-Paque density gradient centrifugation (DGC) causes significant cell loss and influences graft function. The objective of this study was to determine in an animal study whether and how Ficoll-Paque DGC affects the yield and composition of BMNCs compared to alternative isolation methods such as adjusted Percoll DGC or immunomagnetic separation of polymorphonuclear cells (PMNs). Each isolation procedure was confounded by a significant loss of BMNCs that was maximal after Ficoll-Paque DGC, moderate after adjusted Percoll DGC and least after immunomagnetic PMN depletion (25.6±5.8%, 51.5±2.3 and 72.3±6.7% recovery of total BMNCs in lysed bone marrow). Interestingly, proportions of BMNC subpopulations resembled those of lysed bone marrow indicating symmetric BMNC loss independent from the isolation protocol. Hematopoietic stem cell (HSC) content, determined by colony-forming units for granulocytes-macrophages (CFU-GM), was significantly reduced after Ficoll-Paque DGC compared to Percoll DGC and immunomagnetic PMN depletion. Finally, in a proof-of-concept study, we successfully applied the protocol for BMNC isolation by immunodepletion to fresh human bone marrow aspirates. Our findings indicate that the common method to isolate BMNCs in both preclinical and clinical research can be considerably improved by replacing Ficoll-Paque DGC with adapted Percoll DGC, or particularly by immunodepletion of PMNs.
Insights
Ficoll-Paque density gradient centrifugation (DGC) significantly reduces bone marrow mononuclear cell (BMNC) yield and hematopoietic stem cell (HSC) content. Alternative methods like Percoll DGC or immunodepletion offer improved BMNC isolation for regenerative medicine.
Area of Science:
- Cell Biology
- Regenerative Medicine
- Hematology
Background:
- Bone marrow mononuclear cells (BMNCs) are crucial for regenerative medicine.
- Current isolation methods, particularly Ficoll-Paque density gradient centrifugation (DGC), may compromise BMNC yield and function.
- Understanding isolation impacts is vital for optimizing cell-based therapies.
Purpose of the Study:
- To evaluate the effect of Ficoll-Paque DGC on BMNC yield and composition.
- To compare Ficoll-Paque DGC with alternative isolation methods: adjusted Percoll DGC and immunomagnetic depletion of polymorphonuclear cells (PMNs).
- To assess the impact on hematopoietic stem cell (HSC) content.
Main Methods:
- BMNCs were isolated using Ficoll-Paque DGC, adjusted Percoll DGC, and immunomagnetic PMN depletion in an animal model.
- BMNC recovery, subpopulation proportions, and colony-forming unit-granulocyte-macrophage (CFU-GM) for HSC content were analyzed.
- A proof-of-concept study applied immunodepletion to human bone marrow aspirates.
Main Results:
- Ficoll-Paque DGC resulted in maximal BMNC loss (lowest recovery: 25.6±5.8%).
- Adjusted Percoll DGC and immunomagnetic PMN depletion showed significantly higher BMNC recovery (51.5±2.3% and 72.3±6.7%, respectively).
- Hematopoietic stem cell (HSC) content was significantly reduced after Ficoll-Paque DGC compared to the other methods.
Conclusions:
- Ficoll-Paque DGC significantly diminishes BMNC yield and HSC content.
- Adjusted Percoll DGC and immunomagnetic PMN depletion are superior alternatives for isolating functional BMNCs.
- Optimizing BMNC isolation protocols is essential for advancing preclinical and clinical regenerative medicine applications.
