和无血清收集无饲养小鼠胚胎干细胞条件培养基的无细胞方法
Summary
这个协议提供了小鼠胚胎干细胞的血清衍生的集合（MESC）-conditioned培养基（MESC-CM）的方法（胎牛血清，FBS） – 和馈线（小鼠胚胎成纤维细胞，MEF中）用于小区-free条件 – 免费的做法。它可以是适用于老化和老化有关的疾病的治疗。
Abstract
The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.
Introduction
这个协议的目标是从无血清和无饲养培养条件收集小鼠胚胎干细胞（MESC）-conditioned培养基（MESC-CM）和表征其生物学功能。
在一般情况下，胚胎干细胞（ESC）由于它们的多能性和产能自我更新1-3具有用于再生医学，细胞疗法的巨大潜力。然而，干细胞直接移植有几个限制，如免疫排斥和肿瘤形成4,5。因此，无细胞的方法可提供用于再生医学和老化干预6,7-替代的治疗策略。
衰老被视为蜂窝对口组织器官的老化，其特点是生长停滞，改变细胞的生理和行为的永久状态。老化为流行的疾病，包括癌症，心血管疾病，叔的主要危险因素YPE 2型糖尿病，和神经变性8。一个老龄化的明显特征是组织的再生潜能，这是由干细胞衰老和疲惫9造成的下降。许多显著研究显示药理分子，如雷帕霉素9，白藜芦醇10，和二甲双胍11，和血源性系统性的因素，即GDF11 12中，具有一致延缓衰老和延长寿命的能力。
在本研究中，卓制-CM已经收获无血清（胎牛血清，FBS）和馈线（小鼠胚胎成纤维细胞，MEF中）的层以排除血清因子和由MEF分泌因子的污染。这些条件允许的无血清和无饲养的CM那因此启用的MESC特异性分泌因子的准确识别。
这项拟议的协议效率高，相对成本效益，易于进行操作。该技术提供了见解的MESC衍生的可溶性因子表征可以介导的抗衰老作用，其可用于向干预一个安全的和潜在有利无细胞的治疗方法的开发对于衰老相关疾病和其它再生治疗。
Protocol
Representative Results
Discussion
对于无血清和MESC-CM无饲养成功集合，下面的建议，应考虑。最关键的因素是使用早期通道mESCs为MESC-CM的集合。以前，已经显示，相比于晚期通道mESCs早期传代MESC-CM具有更好的抗衰老作用。 mESCs的通道数目已经报道影响其发育潜力16和多能性17。
而分析MESC分泌蛋白质，从而诱发抗衰老效应的具体因素，需要更多的研究，我们目前可以断定MESC-CM足以在细胞水平降…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究是由基础科学研究发展计划（2013R1A1A2060930）和医学研究中心计划（2015R1A5A2009124）通过韩国国家研究基金会（NRF），通过科学，信息和通信技术部提供资金，以及未来规划的支持。这项研究也从病童医院（HK宋）初创工作资助。我们要感谢劳拉·巴维尔和萨拉金JS他们在编辑这个手稿和安德拉斯·纳吉博士提供G4卓制线出色的帮助。
Materials
| DMEM | Invitrogen | #11960-044 | |
| FBS | Invitrogen | #30044333 | 20%, ES cell quality |
| Penicillin and streptomycin | Invitrogen | #15140 | 50units/ml penicillin and 50mg/ml strepto |
| -mycin. | |||
| L-glutamine | Invitrogen | #25030 | 2mM |
| Nonessential amino acids (NEAA) | Invitrogen | #11140 | 100uM |
| β-mercaptoethanol | Sigma | #M3148 | 100uM |
| Leukemia inhibitory factor | Millipore | #ESG1107 | 100units/ml |
| OPTI-MEM | Invitrogen | #22600 | |
| X-gal | Sigma | #B4252 | 1mg/ml |
| Paraformaldehyde (PFA) | Sigma | P6148 | 3.70% |
| Dimethylformamide (DMF) | Sigma | #D4551 | |
| Potassium ferricyanide | Aldrich | #455946 | 5mM |
| potassium ferrocyanide | Aldrich | #455989 | 5mM |
| NaCl | Sigma | #S7653 | 150mM |
| MgCl2 | Sigma | #M2393 | 2mM |
| Mytomycin C | Sigma | #M4287 | 10ug/ml |
| Propidium iodide | Sigma | #P4170 | 50ug/ml |
| TRIzol | Ambion | #15596018 | |
| M-MLV reverse transcript-tase | Promega | #M170B | |
| Power SYBR Green PCR master mix | Applied Biosystems | #4367659 | |
| HDFs, NHDF-Ad-Der-Fibroblast | LONZA | #CC-2511 | |
| Bottle top filter, | Corning | #430513 | 0.2μm |
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