Preparation and immunophenotypic analysis of allo-WJ-MSCs
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EVENTS
At passage 3, when cell cultures reached 80% confluence, adherent cells were detached by treatment with (0.25% trypsin and 2.65 mM EDTA). Live cells were counted using trypan blue staining and a hemocytometer. The release criteria for clinical use of allo-WJ-MSCs comprised the absence of macroscopic clumps, contaminating pathogenic microorganisms, and viability > 95%, with an identity and purity pattern characterized by ≥ 95% positivity for CD73 clone AD2 (PerCP-Cy5.5-conjugated, BD Biosciences TM), CD90 clone 5E10 (FITC-conjugated, eBioscience TM), and CD105 clone MJ7/18 (eFluor450-conjugated, eBioscience TM), and negativity (≤ 2%) for the expression of CD45 clone HI30 (PE-Cyanine 7 conjugated, eBioscience TM), CD34 clone 4H11 (PE-conjugated, eBioscience TM), CD11b clone ICRF44 (APC-conjugated, eBioscience TM), CD14, and Human Leukocyte Antigen-DR isotype (HLA-DR) clone G46-6 (APC-H7-conjugated, BD Biosciences TM). Next, the cell pellet was rinsed once with a staining buffer and resuspended in a loading buffer, and a total of 10,000 events were analyzed per condition. Flow cytometry analysis was performed using an Amnis® CellStream® benchtop system (Luminex, Millipore).The genomic stability of allo-WJ-MSCs was evaluated by karyotyping analysis (data not shown).