Abstract
BackgroundCell transplantation for regenerative medicine has become an appealing therapeutic method; however, stem and progenitor cells are not always freshly available. Cryopreservation offers a way to freeze cells as they are generated, for storage and transport until required for therapy. This study was performed to assess the feasibility of cryopreserving peripheral blood mononuclear cells (PBMCs) for the subsequent in vitro generation of their derived therapeutic population, circulating angiogenic cells (CACs).MethodsPBMCs were isolated from healthy human donors. Freshly isolated cells were either analyzed immediately or cryopreserved in media containing 6% plasma serum and 5% dimethyl sulfoxide. PBMCs were thawed after being frozen for 1 (early thaw) or 28 (late thaw) days and analyzed, or cultured for 4 days to generate CACs. Analysis of the cells consisted of flow cytometry for viability and phenotype, as well as functional assays for their adhesion and migration potential, cytokine secretion, and in vivo angiogenic potential.ResultsThe viability of PBMCs and CACs as well as their adhesion and migration properties did not differ greatly after cryopreservation. Phenotypic changes did occur in PBMCs and to a lesser extent in CACs after freezing; however the potent CD34+VEGFR2+CD133+ population remained unaffected. The derived CACs, while exhibiting changes in inflammatory cytokine secretion, showed no changes in the secretion of important regenerative and chemotactic cytokines, nor in their ability to restore perfusion in ischemic muscle.ConclusionOverall, it appears that changes do occur in cryopreserved PBMCs and their generated CACs; however, the CD34+VEGFR2+CD133+ progenitor population, the secretion of pro-vasculogenic factors, and the in vivo angiogenic potential of CACs remain unaffected by cryopreservation.
Highlights
A unifying definition regarding their characterization does not exist [1,2], endothelial progenitor cells (EPCs) often identified as CD34+VEGFR2+CD133+ cells have the ability to augment postnatal vasculogenesis [3,4,5,6]
Viability of peripheral blood mononuclear cells (PBMCs) but not of Generated circulating angiogenic cells (CACs) is Reduced after Cryopreservation
Fresh samples of PBMCs and CACs showed about 99.760.1% and 95.360.1% viability, respectively
Summary
A unifying definition regarding their characterization does not exist [1,2], endothelial progenitor cells (EPCs) often identified as CD34+VEGFR2+CD133+ cells have the ability to augment postnatal vasculogenesis [3,4,5,6]. EPCs can be generated from the culture of peripheral blood mononuclear cells (PBMCs) isolated from blood by density gradient centrifugation. PBMCs are cultured for 4–7 days in endothelial-promoting media on fibronectin and the subsequently generated therapeutic population is referred to as ‘circulating angiogenic cells’ (CACs), or early EPCs [2,14]. This study was performed to assess the feasibility of cryopreserving peripheral blood mononuclear cells (PBMCs) for the subsequent in vitro generation of their derived therapeutic population, circulating angiogenic cells (CACs)
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