Mesenchymal stromal cell count in bone marrow concentrate is not predicted by nucleated cell count or colony forming units

Abstract

Purpose: Bone marrow aspirate concentrate (BMC) is a point-of-care tool for cartilage repair procedures. It is important to characterize biologics such as BMC both qualitatively and quantitatively so that a successful outcome can be attributed to a specific cellular or bioactive protein composition. Mesenchymal stromal cells (MSCs) are thought to contribute to the effectiveness of BMC, but they are somewhat difficult to quantify and are therefore not commonly characterized in clinical BMC samples. Nucleated cell count (NCC) and colony forming units (CFUs) have been proposed as surrogate markers to quantify MSCs in BMC. The purpose of this study was to test this assumption. We hypothesized that neither NCC nor CFUs would correlate with MSC quantity in BMC. Methods: Bone marrow was aspirated from the sternum of 15 horses (age 13.3±6 years; 4 castrated males, 11 females) and centrifuged to obtain BMC. Complete blood counts were obtained on each BMC sample. Two aliquots of BMC (0.5mL and 1mL) were plated in duplicate and cultured for up to 21 days when CFUs were counted. A CFU was defined as a colony measuring greater than or equal to1mm in diameter assessed by A) an ink stamp on inverted microscopy, or B) visual estimation after crystal violet staining. For each duplicate, one plate was stained with crystal violet, and cells on the other plate were lifted using Accumax (Innovative Cell Technologies, Inc, San Diego), counted, and viability was measured using trypan blue. Based on plastic adherence, these cells were presumed as MSCs and were subjected to confirmatory tri-lineage differentiation assays. A paired t-test was used to compare CFUs counted by inverted microscopy vs crystal violet methods. Bivariate correlation was used to investigate correlation between NCC & MSCs, NCC & CFUs, and CFU & MSCs. Statistical analysis was performed using JMP (SAS Institute, Cary, North Carolina, USA). Significance was set at P < 0.05. Results: Total NCC in BMC samples ranged from 2.5 - 320x103/μl, with a 128-fold variation in NCC, providing a wide range of data for analysis. CFUs were present on 59/68 plates; 9 plates showed no growth. There was no difference in CFUs counts between inverted microscopy and crystal violet methods (p=0.08). Presumptive MSCs ranged from 4 - 60x103 cells/plate. There was no or a very weak correlation between NCC and CFUs (Figure 1a; r=0.08, p=0.38), NCC and MSCs (Figure 1b; r=0.20, p=0.33), and CFUs and MSCs (Figure 1c; r=0.18, p=0.52). Cells were confirmed MSCs (n=10) based on ability to differentiate in adipogenic, osteogenic and chondrogenic lineages. Conclusions: Neither NCC nor CFU are appropriate surrogate methods for quantifying MSCs in BMC. There are several putative bioactive factors in BMC that could be responsible for its therapeutic effect, but to quantify MSC, laboratory-based methods such as tri-lineage differentiation or flow cytometry should be used to characterize BMC.