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

Abstract

In previous studies, vascular smooth muscle progenitor cells (vSMPCs) isolated from peripheral blood mononuclear cells (PBMCs) were cultured using medium containing platelet-derived growth factor-BB (PDGF-BB) for 4 weeks. However, this method requires long culture periods of up to 4 weeks and yields low cell counts. Therefore, we proposed the modified method to improve the cell yield and purity and to reduce the cell culture period. PBMCs were isolated from human peripheral blood and cultured by the conventional method using medium containing PDGF-BB alone or the modified method using medium containing PDGF-BB, basic fibroblast growth factor (bFGF), and insulin-transferrin-selenium ITS for 4 weeks. The purity of vSMPCs was analyzed for the expression of a- smooth muscle actin (SMA) by flow cytometry and significantly higher in the modified method than conventional methods at the 1st and 2nd weeks. Also, mRNA expression of a-SMA by real-time PCR was significantly higher in the modified method than conventional method at the 2 weeks. The yield of vSMPCs by trypan blue exclusion assay was significantly higher in the modified method than conventional method at the 1st, 2nd and 3rd weeks. The primary culture using the modified method with PDGF-BB, bFGF, and ITS 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.

Keywords: Cell culture; PBMCs; PDGF-BB; Peripheral blood; VSMPCs.

Fig. 1

Experimental scheme of vSMPCs culture. vSMPCs was cultured in the modified method and the conventional method for 4 weeks at 1 week of intervals, respectively

Fig. 2

The purity of vSMPCs measured with α-SMA expression by FACS analysis. α-SMA positive cell in HASMCs were measured as a positive control. All data are expressed as mean ± SD. †P < 0.05, Modified method vs. Conventional method. The experiments were performed in triplicate

Fig. 3

mRNA expression of α-SMA in vSMPCs at 2 weeks measured by real-time PCR. mRNA expression of α-SMA in HASMCs was measured as a positive control. The transcripts levels were normalized by comparison with GAPDH. All data are expressed as mean ± SD. †P < 0.05, Conventional method vs. Modified method. The experiments were performed in triplicate

Fig. 4

The yield of vSMPCs assessed by trypan blue exclusion assay. All data are expressed as mean ± SD. †P < 0.05, Modified method vs. Conventional method. The experiments were performed in triplicate

Fig. 5

Morphologic appearance and phenotype of vSMPCs observed by microscope. (a, b) Hill-and-valley morphology. (c, d) α-SMA, (e, f) smMHC, (g, h) CD31 of immunofluorescence (green). (i, j) Result of quantitative analysis of smooth muscle cells specific marker α-SMA and smMHC. CD31 of endothelial surface markers were stained as a negative control. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar: 200 μm (a, b); 100 μm (c–h, in the insets of a, b); 50 μm (in the insets of c–h). The experiments were performed in triplicate