建立小鼠胚胎神经干细胞培养神经球含量
Summary
这个视频协议演示应用程序的神经球实验胚胎每天14鼠脑的神经节金榜，神经干细胞的分离和扩张。
Abstract
在哺乳类动物中，干细胞作为一种未分化的细胞来源，动物的寿命期间保持在不同组织和器官的细胞起源和重建。他们有可能取代细胞在衰老过程中或在损伤和疾病的的过程中丢失。神经干细胞（NSCs）的存在，最初是由雷诺和Weiss（1992），在成年哺乳动物的中枢神经系统使用一种新型的无血清培养系统，神经球检测（NSA）（中枢神经系统）。使用这个实验中，它也是可行的隔离和扩大来自胚胎中枢神经系统的不同地区的神经干细胞。这些在体外培养扩增神经干细胞是多能的，可以引起中枢神经系统的三个主要类型的细胞。虽然NSA似乎相对简单的执行，注意这里展示的程序是必需的，以实现可靠的和一致的结果。实际上该视频演示NSA的产生和扩大神经干细胞从胚胎一天14鼠脑组织和提供技术细节，因此可以实现重复性的神经球文化。过程包括收获，E14小鼠胚胎脑显微切割收获神经节金榜，在国科会中期收获的组织分解，从而获得单细胞悬液，并终于在NSA的文化电镀细胞。经过5-7天的文化，由此产生的初级神经球传代，以进一步扩大，为今后的实验中的神经干细胞的数量。
Protocol
Discussion
神经球检测神经干细胞，因为它的简单性和可重复性1-5隔离和扩张的首选方法。这个实验是一种未分化的中枢神经系统的前体细胞，这可能是在体外和体内研究的大型代的宝贵工具。应该强调的，可以从无论是真正的神经干细胞和更受限制的祖细胞生成神经球。因此，计算神经球的形成频率，简单地高估了真正的神经干细胞在任何特定的神经细胞人口6。要估计真正的神经?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是从Overstreet基金会的资金支持。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| NeuroCult NSC Basal Medium | Medium | Stem Cell Technologies | 05700 | |
| NeuroCult NSC Proliferation Supplements | Medium supplement | Stem Cell Technologies | 05701 | |
| %0.05 trypsin-EDTA | Reagent | Gibco | 25300-062 | |
| Soybean trypsin inhibitor | Reagent | Sigma | T6522 | |
| Pen/Strep | Reagent | Gibco | 15140-122 | |
| *MEM | Reagent | Gibco | 41500-018 | HEM component |
| *HEPES | Reagent | Sigma | H4034 | HEM component |
| *Distilled water | Reagent | Gibco | 15230-147 | |
| Cell strainer | Sieve | BD Falcon | 352340 | |
| T25 flask | Culture ware | Nalge Nunc international | 136196 | |
| T80 flask | Culture ware | Nalge Nunc international | 178905 | |
| 15 ml tubes | Culture ware | BD Falcon | 352096 | |
| 50 ml tubes | Culture ware | BD Falcon | 352070 | |
| Fine curved forceps | Surgical tools | Fine Science Tools | 11251‐35 | |
| Small fine forceps | Surgical tools | Fine Science Tools | 11272‐30 | |
| Small forceps | Surgical tools | Fine Science Tools | 11050‐10 | |
| Fine scissors | Surgical tools | World Percision Instruments, Inc. | 500216 | |
| EGF | Growth factor | R&D | 2028-EG | |
| b-FGF | Growth factor | R&D | 3139-FB | |
| Heparin | Growth factor | Sigma | H4784 | Reconstituted in PBS |
*To make HEM, mix 1×10L packet of MEM and160ml of 1M HEPES and bring the volume to 8.75 L using distilled water. Set the final PH to 7.4 and store it at 4°C.
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