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.
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.
Authors: Sowmya Viswanathan; Armand Keating; Robert Deans; Peiman Hematti; Darwin Prockop; David F Stroncek; Glyn Stacey; Dan J Weiss; Christopher Mason; Mahendra S Rao Journal: Stem Cells Dev Date: 2014-03-10 Impact factor: 3.272
Authors: Thomas G Baboolal; Sally A Boxall; Yasser M El-Sherbiny; Timothy A Moseley; Richard J Cuthbert; Peter V Giannoudis; Dennis McGonagle; Elena Jones Journal: Regen Med Date: 2014-03-12 Impact factor: 3.806