Computational protocol: Comparative Analysis of Cellular and Growth Factor Composition in Bone Marrow Aspirate Concentrate and Platelet-Rich Plasma

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Protocol publication

[…] To determine the presence and proportion of progenitor cells in BMA and BMAC, samples from each patient were evaluated with the CFU-F assay, as previously described [, , ]. The samples (100 μl) were washed twice with phosphate-buffered saline and then resuspended in 3 ml of growth medium composed of Dulbecco's Modified Eagle's Medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and antibiotic–antimycotic solution (Gibco). The cells were seeded in 60 cm2 dishes and cultured at 37°C in a humidified atmosphere of 5% CO2. The medium was replaced 2 days later and nonadherent cells were removed; thereafter, the medium was replaced twice a week. After 2 weeks, the medium was removed and cells were stained with 0.5% Crystal Violet (Sigma-Aldrich) in methanol for 5 min and washed twice with distilled water before the number of CFU-Fs was counted. Colonies of <2 mm in diameter and those that were only faintly stained were excluded. The number of progenitor cells is expressed as the number of CFU-Fs per 106 nucleated cells.In order to analyze the cellular composition of BMAC, the number of CD34+ cells, which are precursors of hematopoietic cells, and CD31−45−90+105+ cells were determined. BM cells in BMAC were incubated in red blood cell lysis buffer (VersaLyse; Beckman Coulter, Brea, CA, USA) at 37°C for 5 min, followed by incubation for 30 min at 4°C with fluorescein isothiocyanate-conjugated anticluster of differentiation (CD)31 (1 : 40), phycoerythrin (PE)/Cy5-conjugated anti-CD90 (1 : 200), energy-coupled dye (PE-Texas Red)-conjugated anti-CD45 (1 : 40), and allophycocyanin- (APC-) conjugated anti-CD105 (1 : 200) antibodies (all from BD Pharmingen, San Jose, CA, USA), and APC-conjugated anti-CD34 antibody (1 : 200; Biolegend, San Diego, CA, USA). Matching isotype control antibodies were used as a control. After incubation, labeled cells were sorted on a Gallios flow cytometer (Beckman Coulter), and data were analyzed using Kaluza software (Beckman Coulter). [...] The numbers of nucleated cells and platelets in BMAC and PRP before and after the concentration procedure were analyzed using Wilcoxon's rank sum tests. The GF quantification results for BMAC and PRP were analyzed using Mann–Whitney U tests. P < 0.05 was considered statistically significant. Analyses were performed using IBM SPSS Statistics v.24.0 (IBM Corp., Armonk, NY, USA). […]

Pipeline specifications

Software tools Kaluza, SPSS
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens