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Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs
Published on: June 17, 2016
Isolation of human mesenchymal stromal cells is more efficient by red blood cell lysis
1Department of Medicine V, University of Heidelberg, Heidelberg, Germany.
Background:
Human mesenchymal stromal cells (MSC) have raised high hopes for tissue engineering and clinical therapy. Their isolation usually involves density fractionation of mononuclear cells (MNC) but this is difficult to standardize, especially under good manufacturing practice (GMP) conditions. MSC represent a heterogeneous mixture of cell types and the composition of subpopulations is affected by the initial steps of cell preparation.
Methods:
This study describes a straightforward method for isolation of human MSC based on red blood cell (RBC) lysis with ammonium chloride. Colony formation was compared directly with Ficoll density fractionation and culture of an untreated whole bone marrow (BM) aspirate.
Results:
After 7 days the number of fibroblastic colony-forming units (CFU-F) per milliliter of BM aspirate was slightly higher upon RBC lysis and the colonies were significantly larger compared with density fractionation, possibly because of maintenance of platelets. In contrast, colony formation was much lower in untreated BM. The heterogeneous composition of subpopulations was reflected by differences between the initial colonies with regard to growth pattern (tight or disperse) and cell morphology (round or elongated). This heterogeneous composition was not affected by the three different isolation methods. Furthermore, enrichment of CD271(+) cells resulted in the same morphologic heterogeneity. All cell preparations demonstrated the same immunophenotype using a panel of surface markers and displayed adipogenic and osteogenic differentiation potential.
Discussion:
This study demonstrates that human MSC can be efficiently isolated by RBC lysis. This technique is faster and can be standardized more easily for clinical application of MSC.
Insights
Red blood cell (RBC) lysis offers a faster, more standardized method for isolating human mesenchymal stromal cells (MSC) for clinical applications. This technique yields more robust colony formation compared to traditional density fractionation.
Area of Science:
- Cell biology
- Tissue engineering
- Regenerative medicine
Background:
- Human mesenchymal stromal cells (MSC) are crucial for tissue engineering and therapy.
- Current isolation methods like density fractionation are difficult to standardize under GMP conditions.
- MSC are heterogeneous, and preparation methods impact subpopulation composition.
Purpose of the Study:
- To describe a straightforward method for human MSC isolation using red blood cell (RBC) lysis.
- To compare RBC lysis with Ficoll density fractionation and untreated bone marrow (BM) culture.
Main Methods:
- Isolation of human MSC via ammonium chloride-induced RBC lysis.
- Comparison of colony formation (CFU-F) with Ficoll density fractionation and untreated BM.
- Analysis of cell subpopulation heterogeneity and differentiation potential.
Main Results:
- RBC lysis yielded slightly more and significantly larger fibroblastic colony-forming units (CFU-F) compared to density fractionation.
- Untreated BM showed significantly lower colony formation.
- Cellular heterogeneity was consistent across all isolation methods, including CD271(+) enrichment.
Conclusions:
- RBC lysis is an efficient method for isolating human MSC.
- This technique is faster and more easily standardized for clinical applications of MSC.
