A new method for neonatal rat ventricular myocyte purification using superparamagnetic iron oxide particles
A new method for neonatal rat ventricular myocyte purification using superparamagnetic iron oxide particles
复制标题
超顺磁性氧化铁颗粒纯化新生大鼠心室肌细胞的新方法
DOI：
10.1016/j.ijcard.2018.05.133
复制
发表时间：
2018-11-01
影响因子：
3.5
通讯作者：
Wang，Huadong
中科院分区：
文献类型：
--
作者：
Yang，Duomeng;Xi，Junmin;Wang，Huadong
新生大鼠心室肌细胞(NRVMs)已被证明是研究心脏疾病的理想模型。然而，目前纯化NRVMs的方法在获得高纯度方面存在局限性。本研究的目的是建立一种利用超顺磁性氧化铁颗粒(SIOP)纯化NRVM的方法。差异附着加或不加溴脱氧尿嘧啶核苷(BrdU)处理组分别为对照组和BrdU组。采用Percoll梯度法(Percoll)和磁激活细胞分选法(MACS)比较SIOP法和SIOP法培养的NRVm的纯度和活性。结果SIOP组的NRVm纯度达93.9 ± 2.0%，免疫荧光计数(IF)为95.6 ± 1.3%。对照组纯度为71.9 ± 2.9%(FCM法)和66.8 ± 8.9%(IF法)，BrdU组为82.0 ± 1.3%(FCM法)和83.1 ± 2.4%(IF法)。SIOP分离的NRVMs的纯度与Percoll组和MACs组无差异。然而，除SIOP方案外，用这些方法分离的心肌细胞均与心脏内源性肾上腺素能细胞混合。分离后24 h，SIOP纯化的NRVm形态相似，细胞产量、存活率和细胞内钙稳态与其他组无差异。结论首次报道了一种高效、新颖的SIOP纯化NRVMs的方法，有助于促进心肌细胞生物学领域的创新研究。
BackgroundNeonatal 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).MethodsNRVMs 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.ResultsThe 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.ConclusionWe first reported an efficient and novel method to purify NRVMs using SIOP, which may help accelerate innovative research in the field of cardiomyocyte biology.