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Updated: Jul 6, 2026
Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice
Published on: January 6, 2018
Isolation and culture of bone marrow-derived human multipotent stromal cells (hMSCs)
Margaret Wolfe1, Radhika Pochampally, William Swaney
1Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, LA, USA.
Abstract:
We have developed protocols whereby a total of 30-90 x 10(6) hMSCs with an average viability greater than 90% can be produced in a single multilevel Cell Factory from a relatively small (1-3 mL) bone marrow aspirate in 14-20 d. It is possible to generate as many as 5 x 10(8) multipotent stromal cells (MSCs) from a single sample, depending on the number of Cell Factories seeded from the initial isolated hMSCs. Briefly, mononuclear cells are collected from a bone marrow aspirate by density gradient centrifugation. The cells are cultured overnight and the adherent cells are allowed to attach to the flask. Nonadherent cells are removed and the culture expanded for 7-10 d with periodic feeding of the cells. The cells are then harvested and seeded at low density (60-100 cells/cm2) into Nunc Cell Factories. The cells are allowed to expand for an additional 7-10 d, and are then harvested.
Insights
This study presents a new protocol for generating over 30 million human mesenchymal stem cells (hMSCs) with high viability from a small bone marrow sample. This method enables large-scale production of multipotent stromal cells for potential therapeutic applications.
Area of Science:
- Stem Cell Biology
- Regenerative Medicine
- Biotechnology
Background:
- Human mesenchymal stem cells (hMSCs) are crucial for regenerative medicine due to their multipotent differentiation capacity.
- Current methods for large-scale hMSC production are often inefficient and costly, limiting their clinical translation.
- Developing scalable and efficient protocols for hMSC isolation and expansion is essential for therapeutic applications.
Purpose of the Study:
- To develop and optimize protocols for the large-scale production of high-viability human mesenchymal stem cells (hMSCs).
- To establish a reproducible method for generating a significant number of multipotent stromal cells from limited bone marrow aspirates.
- To assess the feasibility of using multilevel Cell Factories for efficient hMSC expansion.
Main Methods:
- Bone marrow aspirates (1-3 mL) were processed to isolate mononuclear cells via density gradient centrifugation.
- Adherent cells were cultured and expanded over 7-10 days with periodic feeding.
- Cells were harvested and re-seeded at low density (60-100 cells/cm2) into Nunc Cell Factories for further expansion (7-10 days).
Main Results:
- A total of 30-90 x 10^6 hMSCs with >90% viability were produced per Cell Factory.
- Up to 5 x 10^8 multipotent stromal cells can be generated from a single bone marrow sample.
- The protocol successfully utilizes multilevel Cell Factories for scalable cell expansion.
Conclusions:
- The developed protocols enable efficient and scalable production of high-viability hMSCs from small bone marrow samples.
- The use of Cell Factories significantly enhances the yield of multipotent stromal cells.
- This method provides a promising platform for generating sufficient hMSCs for clinical applications.
