在干细胞移植在体外模拟缺血/再灌注模型
Summary
我们演示了如何建立一个<em>在体外</em>缺血/再灌注模型，以及如何评价干细胞治疗缺血心肌细胞的影响。
Abstract
干细胞移植协议是1,2,3到临床实践发现他们的方式。获得更好的效果，使协议更强大，并寻找新植入的细胞来源，是在最近的研究 4,5的重点。调查的细胞疗法的有效性是不是一件容易的事，需要新的工具，以探讨在治疗过程中所涉及的机制6。我们设计了一个缺血/再灌注损伤的实验协议，以便在缺血/再灌注损伤，干细胞移植后的细胞连接，甚至亚细胞机制的观察和评估细胞疗法的疗效。 H9C2 cardiomyoblast细胞被放置到细胞培养板7,8。缺血葡萄糖与氧气低于0.5％的水平的培养基中150分钟的模拟。然后，正常的媒体和氧含量重新模拟再灌注。氧糖剥夺后，加入他们的文化派生的标记的人骨间质干细胞骨髓移植治疗受损的细胞。在临床试验中的首选，因为它们是方便，微创手术，易于扩展和自体骨髓间质干细胞。共培养24小时后，细胞染色与钙黄绿素和乙锭-二聚体区分活细胞和死细胞。此设置允许我们调查的使用共焦荧光显微镜间的连接和量化缺血细胞流式细胞仪的存活率。共聚焦显微镜显示，如细胞融合和间碳纳米管形成的两个细胞群的相互作用。流式细胞仪分析显示，3受损的细胞，可以绘制在图表上，并进行统计学分析集群。这些人群可以单独进行调查，并根据这些数据得出的结论可以在模拟的有效性的治疗方法。
Protocol
1。准备H9C2细胞cardiomyoblast H9C2大鼠cardiomyoblasts取自ATCC（德国Wesel的，）和扩展的DMEM含10％胎牛血清，4毫米L -谷氨酰胺，100 U / ml青霉素和100μg/ mL链霉素的高糖（4.5克/升）。 H9C2成肌细胞线是从胚胎大鼠心脏产生的，它是用来作为一个在 8,9两个骨骼肌和心肌的体外模型。 准备H9C2细胞的12孔板： 删除的Petri菜H9C2细胞培养液中。 两次2-…
Discussion
证明协议是在体外培养的方法干细胞治疗心肌梗死，这种模式的优点和缺点，更复杂的问题。显然，这不能反映复杂的（如免疫）期间和之后，心肌梗死发生的事件，但可以集中精力对缺血细胞的补充细胞的直接影响。 H9C2 cardiomyoblasts模拟缺血的影响，高度依赖OGD时间，通过对所使用的细胞数量和O 2浓度。一是必须准确地调整这些参数，并发现某些细胞类型的最佳条件，才能有一个标准?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作是支持OTKA（匈牙利科学研究基金）D45933，T049621，TET（匈牙利科学与技术基金会）A4/04，Arg-17/2006和单仲偕，Bolyai，Öveges奖学金和TÁMOP 4.2.2-08 / 1 / KMR – 2008 – 0004和4.2.1 / B 09/1/KMR-2010-0001。 OTKA 83803。我们想感谢威廉Gesztes提供语音。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| calcein-AM | Molecular Probes | L3224, C3099 | http://www.invitrogen.com |
| ethidium homodimer-2 | Molecular Probes | L3224, E3599 | http://www.invitrogen.com |
| Vybrant DiD | Molecular Probes | V22887 | http://www.invitrogen.com |
Table 1. Reagents.
| Name of the equipment | Company | Comments (optional) |
| PeCon cell incubation system for Zeiss microscopes | PeCon GmbH | www.pecon.biz/ |
Table 2. Equipment.
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Tags
Stem Cell TransplantationIn VitroSimulated Ischemia/reperfusion ModelClinical PracticeRobust ProtocolsNew SourcesImplantable CellsCell TherapiesTreatment Process MechanismsExperimental ProtocolIschemia/reperfusion InjuryStem Cell Transplantation EfficacyH9c2 Cardiomyoblast CellsCell Culture PlatesGlucose Free MediumOxygen LevelsReperfusion SimulationDamaged Cells TreatmentLabeled Human Bone Marrow Derived Mesenchymal Stem CellsCo-cultivationLive And Dead Cells Differentiation
Citazione di questo articolo
Cselenyák, A., Benko, Z., Szepes, M., Kiss, L., Lacza, Z. Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model. J. Vis. Exp. (57), e3575, doi:10.3791/3575 (2011).