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

Title
A modified method for effective primary vascular smooth muscle progenitor cell culture from peripheral blood
Other Titles
말초혈액기원 평활근 전구세포를 효과적으로 획득하기 위한 개선된 배양 방법
Author
Jin-Hee Seong
Alternative Author(s)
성진희
Advisor(s)
김경수
Issue Date
2019. 8
Publisher
Graduate School of Biomedical Science & Engineering, Hanyang University
Degree
Master
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.
URI
http://dcollection.hanyang.ac.kr/common/orgView/000000110672http://repository.hanyang.ac.kr/handle/20.500.11754/109392
Appears in Collections:
GRADUATE SCHOOL OF BIOMEDICAL SCIENCE AND ENGINEERING[S](의생명공학전문대학원) > TRANSLATIONAL MEDICAL SCIENCE(임상의과학과) > Theses (Master)
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