Human mesenchymal stromal cells (MSC) raise high hopes for tissue engineering and therapeutic applications. So far, it is not possible to isolate pure fractions from bone marrow and therefore MSC cell preparations notoriously represent heterogeneous mixtures of different cell types. The composition of subpopulations can already be affected by the initial steps of cell preparation. Usually, isolation of MSC involves density fractionation to separate the mononuclear cells (MNCs) from erythrocytes and granulocytes. However, this method is difficult to standardize especially under GMP conditions. Here, we describe an alternative approach for isolation of human MSC based on red blood cell (RBC) lysis with ammonium chloride. This results in a slightly higher number of fibroblastic colony forming units (CFU-F), whereas morphological analysis of the CFU-F reveals the same heterogeneous composition of MSC cultures indicating that the proportion of subpopulations is not affected by RBC lysis. Immunophenotype (CD73+, CD90+, CD105+, CD31−, CD34−, CD45−), adipogenic, and osteogenic differentiation potential of MSC were also similar with both methods. In conclusion, RBC lysis comprises an efficient method for the isolation of human MSC from bone marrow aspirate. This technique is faster and can be standardized more easily for clinical application of MSC.
Mesenchymal stromal cells Mesenchymal stem cells Isolation Red blood cell lysis Density gradient centrifugation Ammonium chloride CFU-F
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We like to thank Anke Diehlmann for assistance in MSC culture. This work was supported by the German Ministry of Education and Research (BMBF) within the supporting program “cell based regenerative medicine” (START-MSC and CB-HERMES), the German Research Foundation DFG (HO 914/7-1), the Joachim Siebeneicher-Stiftung, Germany, and the Academy of Sciences and Humanities, Heidelberg (WIN-Kolleg) and the Stem Cell Network North Rhine Westphalia.
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