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A4.04 A robust assay for fast quantification of multipotential stromal cells in bone marrow aspirates

Background and objectives Bone marrow (BM) aspirate has been used recently as a source of multipotential stromal cells (MSCs) for regenerative treatments of musculoskeletal diseases such as osteoarthritis.1 However the doses of implanted MSCs remain poorly controlled. The numbersof BM MSCs are very low and widely variable depending on the aspiration technique2,3 and donor-related factors.4 Thus, there is a crucial need for a fast quantification method of BM MSCs that is suitable for use within the limited intraoperative/clinic timeframe. Materials and methods Flow-cytometer, Attune was used to enable automated counting of MSCs in BM aspirates (n = 40, 20 males and 20 females, age range 22–80, average 48 years old). For ‘gating out’ non-nucleated cells, Vybrant Ruby dye was employed. The staining concentration, temperature and time for antibodies against CD45 and CD271 to identify BM MSCs were optimised to reduce the staining time to only 5 min. The MSC numbers obtained by this method were compared to longer flow-cytometer-based method 3 and standard colony forming unit-fibroblast (CFU-F) assay. Results Within a total time of 15–20 min, we have successfully optimised an assay to quantify MSCs in only 100 µl of BM aspirate. This simple method was performed without a need to lyse red cells or to include volumetric counting beads. Importantly, the numbers of MSCs obtained using Attune (median 3871 MSCs/ml, range: 96–20,992) were significantly correlated with the counts obtained using LSRII-based method (median 3889 MSCs/ml, range: 87–20,471, r = 0.94, p < 0.0001) or using CFU-F assay (median 128 colonies/ml, range: 3–900, r = 0.72, p = ﻿0.0004). Conclusions We developed a very fast assay for automated counting of MSCs in BM aspirates. This robust assay is ideal to be applied clinically prior to the use of BM aspirates helping to choose and evaluate the dose of MSCs and to improve the outcome of the regenerative therapies. References Veronesi F, Giavaresi G, Tschon M, ﻿﻿et al. Clinical Use of Bone Marrow, Bone Marrow Concentrate, and Expanded Bone Marrow Mesenchymal Stem Cells in Cartilage Disease. Stem cells Dev. 2013;22:181–192 Hernigou P, Homma Y, Flouzat Lachaniette CH, et al. Benefits of small volume and small syringe for bone marrow aspirations of mesenchymal stem cells. Int Orthop. 2013;37:2279–2287 Cuthbert R, Boxall SA, Tan HB, et al. Single-platform quality control assay to quantify multipotential stromal cells in bone marrow aspirates prior to bulk manufacture or direct therapeutic use. Cytotherapy. 2012;14:431–440 Muschler GF, Nitto H, Boehm CA, et al. Age- and gender-related changes in the cellularity of human bone marrow and the prevalence of osteoblastic progenitors. J Orthop Res. 2001;19:117–125