Improved Method for Isolation of Neonatal Rat Cardiomyocytes with Increased Yield of C-Kit+ Cardiac Progenitor Cells.
Improved Method for Isolation of Neonatal Rat Cardiomyocytes with Increased Yield of C-Kit+ Cardiac Progenitor Cells.
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改进了分离新生大鼠心肌细胞的方法，提高了 C-Kit+ 心脏祖细胞的产量。
DOI：
10.4172/2157-7633.1000305
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Alt E
中科院分区：
文献类型：
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作者：
Rutering J;Ilmer M;Recio A;Coleman M;Vykoukal J;Alt E
细胞疗法代表了治疗心脏病的一种很有前途的新范式，心脏病是工业化世界的主要死亡原因。最近发现的组织驻留c-Kit+心脏祖细胞(CPC)推动了利用这些细胞进行再生干预的科学努力，而心肌细胞的原代培养是研究心脏退化和再生的基本分子机制的常见体外模型。目前的心肌细胞分离方法往往导致细胞产量低，成纤维细胞耗竭不足，从而导致心肌细胞在培养中过度生长。在这个方案中，我们描述了一种改进的分离新生大鼠心肌细胞的方法，该方法还可以提高CPC的产量。温和的酶和机械组织处理技术确保了高细胞数量和活力，而随后的Percoll密度梯度离心法将成纤维细胞降至最低。我们比较了不同酶的优点，发现单独使用胶原酶2可以获得非常高的心肌细胞产量，而应用Matrase™酶混合物可以将c-Kit+CPC的相对产量提高到35%。用这种方法分离的心肌细胞和CPC可能成为研究心脏病和基于细胞的治疗方法的重要细胞来源。
Cell therapy represents a promising new paradigm for treatment of heart disease, a major cause of death in the industrialized world. The recent discovery of tissue resident c-Kit+ cardiac progenitor cells (CPCs) has fueled scientific efforts to exploit these cells therapeutically for regenerative interventions, and primary culture of cardiomyocytes is a common in-vitro model to investigate basic molecular mechanisms underlying cardiac degeneration and regeneration. Current protocols for cardiomyocyte isolation frequently result in low cell yield and insufficient depletion of fibroblasts, which then overgrow the cardiomyocytes in culture. In this protocol we describe an improved method for the isolation of neonatal rat cardiomyocytes that also enables enhanced yields of CPCs. Gentle techniques of enzymatic and mechanical tissue processing ensure high cell numbers and viability, while subsequent Percoll density gradient centrifugation minimizes fibroblasts. We compared the advantages of different enzymes and found that Collagenase 2 alone leads to very high yields of cardiomyocytes, whereas the application of Matrase™ enzyme blend increases the relative yield of c-Kit+ CPCs to up to 35%. Cardiomyocytes and CPCs isolated with this protocol may constitute an important cell source for investigating heart disease as well as cell based therapeutic approaches.