Related Experiment Video
Updated: May 25, 2026
Purification, Expansion, and Flow Cytometry-Based Phenotyping of Mouse Derived Bone Marrow Mesenchymal Stem Cells
Published on: July 11, 2025
Single-platform quality control assay to quantify multipotential stromal cells in bone marrow aspirates prior to bulk
Richard Cuthbert1, Sally A Boxall, Hiang Boon Tan
1Academic Unit of Musculoskeletal Disease, Leeds Institute of Molecular Medicine, University of Leeds, UK.
Background Aims:
The manufacture of multipotential stromal cell (MSC)-based products is costly; therefore, a rapid evaluation of bone marrow (BM) 'quality' with respect to MSC content is desirable. The aim of this study was to develop a rapid single-platform assay to quantify MSC in BM aspirates.
Methods:
Aspirated MSC were enumerated using the CD45(-/low) CD271(bright) phenotype and AccuCheck counting beads and compared with a classic colony-forming unit-fibroblast (CFU-F) assay. The phenotype of CD45(-/low) CD271(bright) cells was defined using a range of MSC (CD73, CD105, CD90) and non-MSC (CD31, CD33, CD34, CD19) markers. The effect of aspirated BM volume on MSC yield was also determined.
Results:
CD45(-/low) CD271(bright) cells had a classic MSC phenotype (CD73(+) CD105(+) CD90(+)). Their numbers correlated positively with CFU-F counted manually (R = 0.81, P < 0.001) or using automatic measurements of surface area occupied by colonies (R = 0.66, P < 0.001). Simultaneous enumeration of CD34(+) cells revealed donor variability ranges compatible with standard International Society of Hematotherapy and Graft Engineering (ISHGE) protocols. Aspirating larger marrow volumes gave a significant several-fold reduction in the frequency of CFU-F and CD45(-/low) CD271(bright) cells per milliliter. Therefore aspirated MSC yields can be maximized through a standardized, low-volume harvesting technique.
Conclusions:
Absolute quantification of CD45(-/low) CD271(bright) cells was found to be a reliable method of predicting CFU-F yields in BM aspirates. This rapid (< 40 min) procedure could be suitable for intra-operative quality control of BM aspirates prior to volume reduction/direct injection in orthopedics. In the production of culture-expanded MSC, this assay could be used to exclude samples containing low numbers of MSC, resulting in improved consistency and quality of manufactured MSC batches.
