A modified method for effective primary vascular smooth muscle progenitor cell culture from peripheral blood

Abstract

A modified method for effective primary vascular smooth muscle progenitor cell culture from peripheral blood

Background: In previous studies, vascular smooth muscle progenitor cells (vSMPCs) were isolated from peripheral blood mononuclear cells (PBMCs) through primary cultures using medium containing platelet-derived growth factor-BB (PDGF-BB) alone. However, this method requires long culture periods of up to 4 weeks and yields low cell counts. In this study, we proposed a modified method for vSMPCs culture using medium containing a cocktail of PDGF-BB, basic fibroblast growth factor (bFGF), and insulin-transferrin-selenium (ITS). Material and Methods: PBMCs were isolated from human peripheral blood and cultured by both the conventional and the modified method for 4 weeks. The medium used in the conventional method contained PDGF-BB alone while that used in the modified method contained a combination of PDGF-BB, bFGF, and ITS. The purity and yield of isolated vSMPCs were compared between the two methods at intervals of 1 week. The purity of vSMPCs was assessed through the measurement of α-smooth muscle actin (SMA)-positive cells by flow cytometry and the quantification of α-SMA expression by real-time PCR. The yields of vSMPCs were assessed by trypan blue exclusion assay. Result: Flow cytometry analyses revealed that the fraction of α-SMA-positive cells was significantly higher in the modified method than in the conventional method at the 1st and 2nd week, but the difference became insignificant at the 3rd and 4th week post cell seeding. The expressions of α-SMA transcripts was also significantly higher in the modified method than in the conventional method at 2 weeks after cell seeding. The trypan blue exclusion assay showed that the abundance of vSMPCs peaked in both methods, and was greater in the modified method than in the conventional method, at 2 weeks post cell seeding. Conclusion: The primary culture using the modified medium containing PDGF-BB, bFGF, and ITS showed better cell purity and yield than that using the conventional medium at 2 weeks post cell seeding. Our modified method not only improved cell purity and yield, but also shortened the culture period, compared to the conventional culture method for vSMPCs. The modified method will be a time-saving and useful tool in various studies related to vascular pathology.