隔离和乳鼠心肌细胞冷冻保存
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
Representative Results
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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