Duomeng Yang1, Junmin Xi1, Yun Xing1, Xiangxu Tang1, Xiaomeng Dai1, Kaiying Li1, Hongmei Li1, Xiuxiu Lv1, Daxiang Lu1, Huadong Wang2. 1. Department of Pathophysiology, Key Laboratory of State Administration of Traditional Chinese Medicine of the People's Republic of China, School of Medicine, Jinan University, Guangzhou 510632, Guangdong, China. 2. Department of Pathophysiology, Key Laboratory of State Administration of Traditional Chinese Medicine of the People's Republic of China, School of Medicine, Jinan University, Guangzhou 510632, Guangdong, China. Electronic address: owanghd@jnu.edu.cn.
Abstract
BACKGROUND: Neonatal rat ventricular myocytes (NRVMs) have proven to be an ideal research model for cardiac disease. However, the current methods to purify NRVMs have a limitation to obtain high purity. The purpose of this study was to develop a NRVM purification method by using superparamagnetic iron oxide particles (SIOP). METHODS: NRVMs were purified by using SIOP (SIOP group). The differential attachment with or without bromodeoxyuridine (BrdU) treatment served as control and BrdU groups, respectively. The Percoll gradient (Percoll) and magnetic-activated cell sorting (MACS) methods were performed to compare the purity and viability of NRVMs with SIOP method. RESULTS: The SIOP group enriched NRVMs up to 93.9 ± 2.0% purity determined by flow cytometry (FCM) and 95.6 ± 1.3% by immunofluorescence count (IF). In contrast, the control group gave purities of 71.9 ± 2.9% (by FCM) and 66.8 ± 8.9% (by IF), and the BrdU group obtained 82.0 ± 1.3% (by FCM) and 83.1 ± 2.4% (by IF). The purity of SIOP-isolated NRVMs was not different from that of Percoll and MACS groups. However, the cardiomyocytes separated by these methods, except SIOP protocol, were mixed with intrinsic cardiac adrenergic cells. NRVMs purified by SIOP shaped the similar three-dimensional morphology, with no difference in cell yield, viability and cytosolic Ca2+ homeostasis at 24 h after isolation compared with NRVMs in other groups. Furthermore, SIOP-purified NRVMs retained the responses to phenylephrine and lipopolysaccharide challenge. CONCLUSION: We first reported an efficient and novel method to purify NRVMs using SIOP, which may help accelerate innovative research in the field of cardiomyocyte biology.
BACKGROUND: Neonatal rat ventricular myocytes (NRVMs) have proven to be an ideal research model for cardiac disease. However, the current methods to purify NRVMs have a limitation to obtain high purity. The purpose of this study was to develop a NRVM purification method by using superparamagnetic iron oxide particles (SIOP). METHODS: NRVMs were purified by using SIOP (SIOP group). The differential attachment with or without bromodeoxyuridine (BrdU) treatment served as control and BrdU groups, respectively. The Percoll gradient (Percoll) and magnetic-activated cell sorting (MACS) methods were performed to compare the purity and viability of NRVMs with SIOP method. RESULTS: The SIOP group enriched NRVMs up to 93.9 ± 2.0% purity determined by flow cytometry (FCM) and 95.6 ± 1.3% by immunofluorescence count (IF). In contrast, the control group gave purities of 71.9 ± 2.9% (by FCM) and 66.8 ± 8.9% (by IF), and the BrdU group obtained 82.0 ± 1.3% (by FCM) and 83.1 ± 2.4% (by IF). The purity of SIOP-isolated NRVMs was not different from that of Percoll and MACS groups. However, the cardiomyocytes separated by these methods, except SIOP protocol, were mixed with intrinsic cardiac adrenergic cells. NRVMs purified by SIOP shaped the similar three-dimensional morphology, with no difference in cell yield, viability and cytosolic Ca2+ homeostasis at 24 h after isolation compared with NRVMs in other groups. Furthermore, SIOP-purified NRVMs retained the responses to phenylephrine and lipopolysaccharide challenge. CONCLUSION: We first reported an efficient and novel method to purify NRVMs using SIOP, which may help accelerate innovative research in the field of cardiomyocyte biology.