人类神经的聚集体cGMP的适用展开法干细胞和祖细胞来源从多能干细胞或胎儿脑组织
Summary
这个协议描述了一种新颖的机械斩波方法，它允许球形神经干细胞和祖细胞的聚集体的膨胀而不分离为单个细胞悬浮液。维持细胞/细胞接触可超过40代快速稳定增长。
Abstract
一个细胞扩展技术聚敛大量的细胞从单个样本进行实验研究和临床试验，将大大有利于干细胞的社区。目前，很多的扩展方法是费力，成本高，并且涉及完整的解离可能会导致几个干细胞和祖细胞类型来进行分化或提早衰老。为了克服这些问题，我们开发了称为“斩波”，即简单又便宜的自动机械传代方法。该技术避免化学或酶分解成单个细胞，而是允许大规模扩张暂停，即保持恒定的细胞/细胞接触球体的文化。斩波方法主要被用于胎儿脑源性神经祖细胞或神经球，并于最近出版了由胚胎和诱导多能干细胞源性神经干细胞的使用。该过程涉及。ES神经球接种到组织培养培养皿中，并随后通过锋利，无菌刀片通过细胞有效地自动化的手动机械解离每个球的繁琐过程。在培养悬浮细胞提供了有利的表面积与体积之比;如超过500,000个细胞可以在小于0.5毫米的直径的单个神经球生长。在一个T175烧瓶中，超过50万个细胞可以相比，只有15万美元的贴壁培养在悬浮培养生长。重要的是，砍程序已经根据现行良好生产规范（cGMP）的使用，允许大规模量产的临床级细胞产品。
Introduction
还有作为一个单层或1-3总神经球4-7在文化扩张啮齿类动物神经干细胞的悠久历史。此外，从显影中枢神经系统8-17的各区域分离的人神经前体细胞（hNPCs）已在体外扩展。这些细胞是双强有力的，能分化成两个星形胶质细胞和神经元，并已在研究神经发育18,19和疾病机制20,21一种非常有用的工具。 hNPCs也被移植到中枢神经系统疾病的许多不同的动物模型中具有不同集成，存活和功能的影响22-24的水平。
传统上，啮齿动物或人胎儿来源的NPC暴露于生长因子-通常表皮生长因子（EGF）和/或成纤维细胞生长因子-2（FGF-2）25-28 -和贴壁29和三个维球体系统一般传代使用酶解成单细胞悬液30-34。扩大细胞研究或临床应用的标准方法是为由于易于操作的单层贴壁。然而，我们已经表明，传代单层和神经球hNPCs用酶或化学溶液导致早期衰老35。此外，酶的解离可能导致分化和异常核型的基础上表现出与胚胎干细胞36-38数据水平增加。虽然传代hNPCs的标准方法制作了现行良好生产规范已经进入了第一阶段的临床试验（干细胞公司，Neuralstem公司）（cGMP）的高档产品，该方法只允许几个回合细胞扩增，限制了银行的潜力。
显然，大型科研实验和未来的临床试验可能会从能力中受益繁殖细胞的体积和具有延迟衰老，允许大规模的生长和细胞银行。为了满足这一需求，我们开发了一种新的和机械的完好传代神经球自动化的方式由“砍”成小群，以维持细胞与细胞之间的接触。这种方法大大增加了它们的使用寿命39和悬浮培养允许一个更有效的利用培养箱空间相比单层培养物，观察用一种替代3D生物反应器培养方法40。所提供的斩波协议允许用于生产大型银行从一个胎样品大于通道10，使用标准的传代方法一个不太可能的技艺。虽然这个方法传代hNPCs是标新立异，它是越来越受欢迎，最近，出版了其他类型的细胞，如人类胚胎和诱导多能干细胞源性神经干细胞，从而实现大规模扩张的各种应用，包括v中ITRO疾病模型41-46。重要的是，cGMP的同类HNPC细胞库已经生产的斩波方法，这表明该技术可以对将来的临床应用被应用。
Protocol
1，道德声明和安全此过程涉及使用从人类或动物来源的细胞培养产品。所有派生的组织必须通过适当的机构审查委员会（S）和/或机构动物护理和使用委员会（S）使用前必须获得批准。 所有的生物危险废物必须按照到时由相应的机构决定的安全法规。了解并遵守所有的贴切安全和处置指导整个过程。 2，准备设备，耗材，试剂，并观察准备?…
Representative Results
hNPCs冻结在P19的图5。代表性的数据A）投影手机号码，然后解冻，用酶解相比，神经球经斩波方法传代扩大为单层贴壁。 0天表示，当细胞解冻，在P20 B）领域的代表图像的前印章，球后的印章，10X D）色样用来描述在媒体调理程度的cGMP的10倍。 <stron…
Discussion
图6。斩波电路图 。使用机械斩波方法，培养扩大球体干/祖细胞。
关键步骤
斩波扩张模式的概况示于图6。HNPC球的大小是传代神经球之前，观察重要的标准之一。虽然有在球体大小的大方差，球体的至少30％应具有的直…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Soshana斯文森博士的严格审查及本报告的编辑。这项工作是由NIH / NINDS 1U24NS078370-01和CIRM DR2A-05320做出了贡献。
Materials
| Beaker, 50 mL | Fisherbrand | FB-100-50 | multiple manufacturers/suppliers |
| Bio-Safety Cabinet, class II | Baker | SG-603A | 4 ft. or 6 ft. model. 6 ft. model recommended; multiple manufacturers/suppliers |
| Blades, Double-edge Prep | Personna | 74-0002 | multiple manufacturers/suppliers. CAUTION: Sharp |
| Cell Freezing Media | Sigma-Aldrich | C6295-50ML | DMSO, serum-free |
| Centrifuge, swing-bucket with 15 mL inserts | Eppendorf | 5810 R | multiple manufacturers/suppliers |
| Conical Tubes, 15 mL | Fisherbrand | S50712 | multiple manufacturers/suppliers |
| Conical Tubes, 50 mL | BD Falcon | 352074 | multiple manufacturers/suppliers |
| Controlled Rate Freezer | Planer | Kryo 750 | multiple manufacturers/suppliers |
| Cryovials, 2 mL | Corning | 430488 | multiple manufacturers/suppliers |
| Culture Flask, Vented, T12.5 | BD Falcon | 353107 | multiple manufacturers/suppliers |
| Culture Flask, Vented, T25 | BD Falcon | 353081 | multiple manufacturers/suppliers |
| Culture Flask, Vented, T175 | BD Falcon | 353045 | multiple manufacturers/suppliers |
| Culture Flask, Vented, T75 | BD Falcon | 353110 | multiple manufacturers/suppliers |
| Filter, 0.22 µm, attached cup, 1 L | Millipore | SCGPU11RE | multiple manufacturers/suppliers |
| Filter, 0.22 µm, attached cup, 150 mL | Millipore | SCGVU01RE | multiple manufacturers/suppliers |
| Filter, 0.22 µm, attached cup, 500 mL | Millipore | SCGPU05RE | multiple manufacturers/suppliers |
| Filter, 0.22 µm, attached cup, 50 mL | Millipore | SCGP00525 | multiple manufacturers/suppliers |
| Filter Paper, 8.5 cm circles | Whatman/GE | 1001-085 | |
| Forceps, Standard Pattern – Serrated/Curved/18 cm | Fine Science Tools | 11001-18 | |
| Freezing Chamber, Isopropyl Alcohol | Nalgene | 5100-0001 | "Mr. Frosty" |
| Incubator, 37°C/5% CO2 | Forma | 370 series | multiple manufacturers/suppliers |
| Hemacytometer, Phase | Hausser Scientific | 1475 | multiple manufacturers/suppliers |
| McIlwain Tissue Chopper | Lafayette Instruments | TC752-PD | Petri dish modification required. CAUTION: Moving, sharp blade. |
| Micropipettor, 1 – 10 μL | Gilson | F144562 | multiple manufacturers/suppliers |
| Micropipettor, 100 – 1000 μL (starter kit) | Gilson | F167700 | multiple manufacturers/suppliers |
| Micropipettor, 2 – 20 μL (starter kit) | Gilson | F167700 | multiple manufacturers/suppliers |
| Micropipettor, 20 – 200 μL (starter kit) | Gilson | F167700 | multiple manufacturers/suppliers |
| Nutdriver, Autoclavable, 5/16" | Steritool | 10302 | |
| Pasteur Pipets, cotton-plugged | Fisherbrand | 13-678-8B | multiple manufacturers/suppliers |
| Petri Dish, Glass, Autoclavable | Corning | 3160-100 | |
| Pipet Aid | Drummond | 4-000-101 | multiple manufacturers/suppliers |
| Shim disc | McMaster-Carr | VARIABLE | multiple manufacturers/suppliers |
| Sterile barrier pipet tips, 10 μL | AvantGuard | AV10R-H | multiple manufacturers/suppliers |
| Sterile barrier pipet tips, 1000 μL | AvantGuard | AV1000 | multiple manufacturers/suppliers |
| Sterile barrier pipet tips, 20 μL | AvantGuard | AV20-H | multiple manufacturers/suppliers |
| Sterile barrier pipet tips, 200 μL | AvantGuard | AV200-H | multiple manufacturers/suppliers |
| Sterile Disposable pipettes, all-plastic wrap, 10 mL | Fisherbrand | 13-676-10J | multiple manufacturers/suppliers |
| Sterile Disposable pipettes, all-plastic wrap, 2 mL | Fisherbrand | 13-675-3C | multiple manufacturers/suppliers |
| Sterile Disposable pipettes, all-plastic wrap, 25 mL | Fisherbrand | 13-676-10K | multiple manufacturers/suppliers |
| Sterile Disposable pipettes, all-plastic wrap, 5 mL | Fisherbrand | 13-676-10H | multiple manufacturers/suppliers |
| Sterilization Pouches, 19 x 33 cm | Crosstex | SCL | multiple manufacturers/suppliers |
| Strainer, 40 µm | BD Falcon | 352340 | |
| Tissue Culture Dishes, 60 mm | BD Falcon | 351007 | |
| Tube Racks, Interlocking Four-Way | Fisherbrand | 03-448-17 | |
| Water Bath | Fisherbrand | S52602Q | multiple manufacturers/suppliers |
| Neural Progenitor Cell-Specific Processing Reagents | |||
| Neural Stem Cell Expansion Medium (Stemline) | Sigma-Aldrich | S3194-500ML | Important to use the Stemline brand |
| Recombinant Human Epidermal Growth Factor (EGF) | Millipore | GF316 | multiple manufacturers/suppliers |
| Recombinant Human Leukemia Inhibitory Factor (LIF) | Millipore | LIF1010 | multiple manufacturers/suppliers |
| Trypan Blue (0.4%) | Sigma-Aldrich | T8154-100ML | multiple manufacturers/suppliers |
| TrypLE Select (1X) | Life Technologies | 12563-011 |
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Cite This Article
Shelley, B. C., Gowing, G., Svendsen, C. N. A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue. J. Vis. Exp. (88), e51219, doi:10.3791/51219 (2014).