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Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells
Published on: May 31, 2024
High-yield isolation, expansion, and differentiation of rat bone marrow-derived mesenchymal stem cells with fibrin
Lior Zangi1, Rachel Rivkin, Ibrahim Kassis
1Biotechnology and Radiobiology Laboratory, Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
Abstract:
Fibrin microbeads (FMB), made of extensively cross-linked dense and partially denatured fibrin, were used as a matrix for efficient isolation of mesenchymal stem cells (MSC) from rat bone marrow (BM). After 2 days of incubation of FMB with whole BM in suspension, a high number of cells of mesenchymal origin attached to the FMB. On the 14th day after their transfer to plastic, the yield of the cells isolated via FMB was approximately 3-4 times higher than that obtained by currently used protocols based solely on plastic adhesion. This implies that the number of MSC in BM may be higher than previously reported. FACS analyses and immunostaining showed the mesenchymal characteristics of these cells by positive staining for fibronectin, vimentin, CD49E, and CD29. Immediately after isolation, less than 20% of the cells still expressed the hematopoietic markers CD11b and CD45. Most of these cells were eventually eliminated after further expansion of the isolated cells on plastic. Cells isolated via FMB were expanded in culture for more than 4 months and could be defined as MSC along this time period based on their ability to differentiate into precursors of mesenchymal tissues, such as osteogenic, adipogenic, and chondrogenic cells. Similar differentiation plasticity was observed in clones derived from single cells from whole MSC populations isolated via FMB. Based on our results we propose that FMB can serve as a 3-dimensional biodegradable matrix for isolation, differentiation, and possibly implantation of MSC for tissue regeneration.
Insights
Fibrin microbeads efficiently isolate mesenchymal stem cells (MSC) from rat bone marrow, yielding 3-4 times more cells than traditional methods. These MSCs maintain their characteristics and differentiation potential for extended culture periods.
Area of Science:
- Biomaterials Science
- Stem Cell Biology
- Regenerative Medicine
Background:
- Mesenchymal stem cells (MSCs) are crucial for tissue regeneration.
- Current isolation protocols for MSCs from bone marrow have limitations.
- Efficient and higher-yield isolation methods are needed.
Purpose of the Study:
- To evaluate fibrin microbeads (FMB) as a novel matrix for MSC isolation from rat bone marrow.
- To compare the yield and characteristics of MSCs isolated using FMB versus traditional plastic adhesion.
- To assess the long-term culture potential and differentiation capacity of FMB-isolated MSCs.
Main Methods:
- Rat bone marrow was incubated with FMB in suspension.
- Cell attachment and proliferation on FMB were monitored.
- Flow cytometry (FACS) and immunostaining were used to characterize isolated cells.
- Differentiation potential into osteogenic, adipogenic, and chondrogenic lineages was assessed.
- Clonal analysis was performed on single-cell-derived populations.
Main Results:
- FMB facilitated high attachment of mesenchymal cells within 2 days.
- FMB isolation yielded 3-4 times more MSCs compared to plastic adhesion after 14 days.
- Isolated cells exhibited positive MSC markers (fibronectin, vimentin, CD49E, CD29) and low hematopoietic marker expression.
- FMB-derived MSCs maintained their phenotype and differentiation plasticity for over 4 months in culture.
- Clones derived from FMB-isolated MSCs showed similar differentiation potential.
Conclusions:
- Fibrin microbeads serve as an effective 3D biodegradable matrix for efficient MSC isolation from bone marrow.
- This method significantly increases MSC yield and provides cells with robust differentiation capabilities.
- FMB technology holds promise for MSC isolation, expansion, and potential therapeutic applications in tissue regeneration.
