Abstract
即使很大的进展已在帕金森氏病的临床表征而作出的，一些研究报告，帕金森氏病的诊断没有病理证实在高达临床诊断帕金森氏病的25％。因此，从组织临床确诊患者原发性帕金森病可有误诊率高收取;因此在从这些组织体外研究来研究帕金森氏病作为临床前模型中可以成为徒劳。
通过收集死后人软脑膜与帕金森氏病的一个证实神经病理学诊断和特征在于黑质纹状体细胞损失和细胞内蛋白质的夹杂物称为路易体，人们可以肯定，临床观察帕金森未被另一个潜在的疾病进程（ 例如肿瘤，动脉硬化）引起的。
该协议presenTS解剖，并准备为脑膜成纤维细胞培养的推导死后人类软脑膜的。此过程是健壮和具有高的成功率。培养物的挑战是无菌的脑采购一般不无菌条件下进行。因此，要在培养介质用青霉素，链霉素和两性霉素B的鸡尾酒补充是很重要
脑膜成纤维细胞的尸检确诊病例与帕金森氏症的推导为帕金森氏病的体外模型的基础。脑膜成纤维细胞出现制备样品后3-9天，约20-30亿个细胞可在6-8周冷冻保存。脑膜成纤维细胞培养是同质和细胞表达纤维连接蛋白，一种常用的标记来识别脑膜。
Introduction
脑膜由保护大脑三种细胞膜的：硬脑膜，蛛网膜和软脑膜。最近，已经认识到，脑膜也起到大脑发育和脑稳态1中起重要作用。脑膜从间充质和神经嵴源性细胞衍生和有趣的是，它已被证明存在于脑膜该祖细胞可以产生的神经元在体外和体内 2，3，4移植后。脑膜培养已还成功地用作饲养层，因为它们具有基质细胞衍生诱导活性为胚胎干细胞分化为多巴胺能神经元5。此外，软脑膜必须直接分化成神经元，星形胶质细胞和少突胶质缺血条件6下的可能性。
此协议，人死后的脑膜样本从蛛网膜和软脑膜收集，统称为软脑膜，并且采购作为人脑捐赠用于研究目的的一部分。大脑的解剖是死亡的24小时之内进行，并且如所示在这里这个协议软脑膜样品置于冷生长培养基用于进一步的处理的下一个6-8小时内。
这个协议描述了用于患者主软脑膜细胞培养的发展解剖和制备的人类脑膜样品。将组织切成25-30片大约3mm×3mm的正方形。三片置于每个6孔明胶包好，并与圆形盖玻片按住。脑膜解剖大约需要25-35分钟。这种文化的主要挑战是，一般不会无菌条件下进行无菌为大脑采购，运输和清扫。 ŧherefore，到培养基用青霉素，链霉素和两性霉素B的鸡尾酒补充并使用多孔培养皿分别培养组织片是很重要的。
脑膜成纤维细胞的生长通常在第一个星期内发生。介质每两至三天更换直到细胞汇合，细胞酶促传代。脑膜成纤维细胞是在每毫升/瓶1百万个细胞在冷冻保存介质冻存。有了这个协议，20-30万元脑膜成纤维细胞可在6-8周衍生为冷冻保存。这些脑膜成纤维细胞的下游应用是用于疾病研究，直接的神经元分化，或从软脑膜为疾病机制的理解以及对药物开发诱导多能干细胞的衍生初级培养物。
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Materials
| Name | Company | Catalog Number | Comments |
| Corning Petri dishes | Fisher Scientific | 351029 | |
| Nunc 6-well plate | Fisher Scientific | 14-832-11 | |
| 15-mm cover slips | Fisher Scientific | 12-545-83 15CIR-1D | |
| Scalpels, sterile blade, No. 15 | Miltex | 4-415 | |
| Curved precision tip forceps | Fisher Scientific | 16-100-122 | |
| Serological pipettes | Fisher Scientific | 13-678-11E | |
| Pasteur pipettes | Fisher Scientific | 22-230490 | |
| Gelatin | Sigma | G1890-100G | |
| Phosphate Buffer Saline | Fisher Scientific | SH30264.02 | |
| Corning 500 mL filter unit | Fisher Scientific | 430770 | Combine media components and filter. |
| Nunc Cell Culture Treated Flasks with Filter Caps, T175 cm2 | Thermo Scientific | 178883 | |
| Name | Company | Catalog Number | Comments |
| Growth Media | |||
| Hyclone DMEM | Fisher Scientific | SH30081.02 | |
| Hyclone FBS | Fisher Scientific | SH30910.03 | |
| MEM Non-Essential Amino Acids Solution (100x) | Thermo Fisher | 11140-050 | |
| GlutaMAX Supplement (100x) | Thermo Fisher | 35050-061 | |
| Sodium Pyruvate (100 mM) | Thermo Fisher | 11360-070 | |
| Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher | 15140-122 | |
| Amphotericin B (Yellow Solution/250 µg/mL) | Fisher Scientific | BP264520 | |
| Bambanker Freeze 120 mL | Fisher Scientific | NC9582225 | |
| Name | Company | Catalog Number | Comments |
| Fibronectin Staining | |||
| 8 well chamber slides | Fisher Scientific | 1256518 | |
| 20% paraformaldehyde | Electron Microscopy Sciences | 15713 | |
| Triton X-100 | Sigma | T8787 | |
| 100% Glycerol | BioRad | 9455 | |
| 100% normal goat serum | Fisher Scientific | 101098-382 | |
| Anti-Fibronectin antibody [F1] | Abcam | ab32419 | 1:300 dilution in blocking solution |
| Anti-SERPINH1 | Sigma | S5950-200ul | 1:250 dilution in blocking solution |
| Anti-SOX2 | Millipore | MAB4343 | 1:100 dilution in blocking solution |
| Anti-Nestin | Millipore | MAB5326 | 1:200 dilution in blocking solution |
| Anti-TUJ1 | Covance | MMS-435P | 1:1,000 dilution in blocking solution |
| Alexa Fluor 488 anti-rabbit | Thermo Fisher | A11029 | 1:400 dilution in blocking solution; (green channel; Ex/Em2 495/519 nm) |
| Alexa Fluor 555 anti-mouse | Thermo Fisher | A21424 | 1:400 dilution in blocking solution; (red channel; Ex/Em2 590/617 nm) |
| Hoechst 33342 stain | Thermo Fisher | H3570 | dilute to a final concentration of 1.0 μg/mL; (blue channel; Ex/Em2 358/461 nm) |
| Suppliers are suggestions, similar products from alternative vendors can be used as well. | |||
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