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[Efficient isolation of mesenchymal stem cells from human bone marrow by direct plating method combined with modified
Wen Xing1, Ai-Ming Pang, Jian-Feng Yao
1Chinese Academy of Medical Sciences, Tianjin, China.
Abstract:
Human bone marrow is the major source of mesenchymal stem cells (MSC). It was reported that the standard density gradient centrifugation method was not efficient in isolating MSC and it may be caused by the existing of bone marrow particles. In previous study, a lot of MSC were obtained by culturing bone marrow particles alone combined with standard method. However, it is time- and labor-consuming to obtain bone marrow particles by filtering and to isolate MNC by density gradient centrifugation. This study was purposed to explore the more simple and efficient method to isolate MSC from bone marrow. Seven normal bone marrow aspirates were collected and centrifugated. The bone marrow particles floated on surface layers were cultured by modified primary explant culture, whereas the bone marrow aspirates deposited were cultured by direct plating method, then the immun phenotype and differentiation capability of isolated cells were analyzed. The results showed that in 3 of 7 aspirates, bone marrow particles were floated on surface layers, whereas the other bone marrow cells and some particles were deposited after centrifugation. The MSC were reliably isolated from the floating layers or deposited aspirates by modified primary explant culture and direct plating method separately. After 3 passages the isolated MSC did not express CD45 and CD34, but expressed CD105, CD73, CD44, CD90, CD49e and they could differentiate into chondrocytes and adipocytes. It is concluded that normal human bone marrow MSC can be isolated simply and efficiently by direct plating method in combination with modified primary explant culture.
Insights
This study presents a simpler, efficient method for isolating mesenchymal stem cells (MSCs) from human bone marrow. The direct plating method combined with modified primary explant culture successfully yielded MSCs with desired characteristics.
Area of Science:
- Stem Cell Biology
- Hematology
- Biotechnology
Background:
- Human bone marrow is a primary source of mesenchymal stem cells (MSCs).
- Standard density gradient centrifugation is inefficient for MSC isolation due to bone marrow particles.
- Previous methods involving bone marrow particle isolation are time-consuming.
Purpose of the Study:
- To explore a simpler and more efficient method for isolating MSCs from human bone marrow.
- To evaluate the immunophenotype and differentiation capabilities of MSCs isolated by novel methods.
Main Methods:
- Seven normal human bone marrow aspirates were collected and processed.
- Bone marrow particles were cultured using modified primary explant culture.
- Deposited bone marrow aspirates were cultured using direct plating.
- Immunophenotype and differentiation potential of isolated cells were analyzed.
Main Results:
- MSCs were successfully isolated from both floating bone marrow particles and deposited aspirates.
- Isolated MSCs, after 3 passages, lacked CD45 and CD34 expression.
- Isolated MSCs expressed MSC markers (CD105, CD73, CD44, CD90, CD49e) and differentiated into chondrocytes and adipocytes.
Conclusions:
- A simple and efficient method for isolating human bone marrow MSCs was developed.
- The combination of direct plating and modified primary explant culture is effective.
- This method provides a practical approach for MSC isolation for research and potential therapeutic applications.
