Summary

隔离和乳鼠心肌细胞冷冻保存

Published: April 09, 2015
doi:

Summary

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Abstract

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for α-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

Introduction

心肌细胞培养是现代心脏研究的一个重要工具。乳鼠心肌细胞(新农合),因为隔离常用和文化是比成年大鼠心肌1更容易。该NRCM方法仍然有一些限制,包括长期分离过程和有限的细胞增殖的菜。有对新农合的大多数一般需要4-48小时工作2-6的隔离无数协议。此外,将细胞分离经常由1至2天龄的幼鼠2,4-7;诞生时间是不可预知和冲突,在实验室的其他工作。如果需要对实验只有少量细胞的隔离可能是低效和浪费的。最努力改善工作流程专注于减少隔离时间,但是这并没有解决定时幼崽诞生的问题。

作为替代,许多实验室利用从胚胎干细胞(ESC)或诱导的多能干细胞(iPS细胞)衍生的心肌细胞。然而,重新编程和/或分化的过程可能是非常耗时和昂贵的为好。使用这些细胞作为体外肌细胞模型时,可以有其他的问题。既ESC-和iPSC-衍生的心肌细胞已显示出表现从初级心肌8-10在电生理学差异。

解离合管能够被存储为使用冷冻11几天,然而这并允许长期存储。液氮通常用于保存细胞的时间较长期间,但是需要冷冻保护剂,如二甲基亚砜(DMSO)。此前有研究表明,在冻结媒体5-10%DMSO的理想浓度允许新农合的冷冻保存,但即使是这样的生存能力仍然很低12。虽然DMSO有助于释放过程中保护细胞诚,它可以是对细胞有毒的浓度高于1.5%的13。以前的研究已经表明,慢慢地从细胞中除去DMSO,可提高细胞活力14。

我们试图提高NRCM基于细胞的测定通过冷冻保存的细胞下述分离的效率。这允许了细胞被解冻,并且在必要时,减小隔离和动物消费的频率使用。使用这种方法,我们表明,它是可能的冷冻保存新农合和解冻它们用于在以后的时间。解冻后细胞维持可接受的可行性,并产生NRCM文化是积极的α-横纹肌辅肌动蛋白(α-SA)和合同油然而生。

Protocol

下面的协议是专为心肌细胞的新生幼鼠(10-14幼仔)一窝隔离。如果产仔显著不同,该过程可能需要进行调整以补偿。小狗应该在48±6小时岁。所有的程序在此已批准北卡罗莱纳州立大学的机构动物护理和使用委员会(IACUC)。 1.细胞分离制备注:隔离之前执行以下步骤的一天。 高温高压灭菌以下:大剪刀,小剪刀,小尖镊子,镊子大。使用重力?…

Representative Results

以下的新生大鼠心肌细胞的分离,将细胞冷冻向下液氮,并且可以贮存至少几个月。通常在解冻时,用台盼蓝分析测定的生存能力将介于40-60%。虽然这是比其他类型的细胞时,用适当的接种和培养的细胞将增殖( 图1)。为了验证收缩,细胞可以用相差显微镜进行成像。细胞应开始于约三天( 图2)自发收缩。对于免疫细胞化学(ICC)分析,将细胞接种在4孔培养载玻片?…

Discussion

该协议允许新农合被孤立,冷冻,解冻。解冻的细胞是该过程的一个重要部分。一系列的DMSO稀释液被用来慢慢地从单元14除去DMSO中。重要的是DMSO中提取被快速地执​​行作为所述细胞是特别敏感解冻后立即死亡。如果需要更多或更少的细胞解冻,根据需要的DMSO溶液的体积可以按比例。

一个挑战NRCM隔离和文化是成纤维细胞的快速增殖。该协议使用预镀已显示减少的?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by funding from American Heart Association 12BGIA12040477, NC State University Chancellor’s Faculty Excellence Program, and National Natural Science Foundation of China H020381370216.

Materials

IMDM (+25mM HEPES +L-glutamine) Gibco 12440-046 With added 25mM HEPES and L-glutamine
L-glutamine Gibco 25030-081
FBS Hyclone SH30070.03
Gentamicin Gibco 15710-064
2-Mercaptoethanol Gibco 21985-023
HBSS (+Ca +Mg) Corning 21-020-CV With added calcium and magnesium, pH 7.1-7.4
Trypsin 0.25% Gibco 25300-056
Trypsin 0.05% Gibco 25300-054
Cryostor CS5 BioLife Solutions 205102 Freezing media
Cryogenic Vial Corning 430659
Collagenase Sigma C1889-50MG
40μm Cell Strainer Greiner Bio-One 542040
Sterilizing Vacuum Filter (0.22μm) Corning 431118
50mL Conical Corning 430828
15mL Conical Corning 430790
trypan blue Cellgro 25-900-CI
Mr Frosty Freezing Container ThermoScientific 5100-0001
Millicell EZ SLIDES Millipore PEZGS0416
α-sarcomeric actinin antibody Sigma A7811
Fibronectin Corning 356008
Bromodeoxyuridine BD Biosciences 51-7581KZ

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Cite This Article
Vandergriff, A. C., Hensley, M. T., Cheng, K. Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes. J. Vis. Exp. (98), e52726, doi:10.3791/52726 (2015).

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