一个<em>体外</em鼠类的椎间盘>器官培养模型
Summary
椎间盘（IVD）的整个器官培养保留天然的细胞外基质，细胞的表型，和细胞 – 基质相互作用。在这里我们将介绍使用鼠标腰部，并在其脊柱功能单位尾椎的IVD并利用该系统的几个应用的IVD文化体系。
Abstract
椎间盘（IVD）变性是一个显著贡献腰痛。体外诊断是用于传输和抑制负荷在脊椎关节的纤维软骨。的IVD由富含蛋白聚糖髓核（NP）和通过软骨端板夹在中间的富胶原蛋白的纤维环（AF）的。连同相邻椎骨，椎骨-IVD结构形成功能性脊柱单元（FSU）。这些微结构包含独特的细胞类型，以及独特的细胞外基质。的FSU的整个器官培养保留天然的细胞外基质，细胞分化的表型，和细胞 – 基质相互作用。因此，器官培养技术是用于研究IVD的复杂生物学机制是特别有用的。在这里，我们描述了整个培养腰鼠标FSU的，它为研究疾病机理和治疗的IVD的理想平台的高通量方法。此外，我们描述了小号利用此器官培养方法进行进一步的研究，其中包括对比增强显微CT成像和IVD的三维高分辨率有限元建模everal应用程序。
Introduction
腰痛（LBP）是全球残疾和工作场所的主导因素生产力损失，以及单独的美国人超过50十亿美元花费在治疗腰痛1。虽然普遍，LBP的病因仍然是复杂和多因素。然而，椎间盘（IVD）退变是LBP 2最显著的危险因素之一。
的IVD由三个微观结构：外纤维环（AF）中，内部髓核（NP），以及近端和远端3夹住整个结构的两个端板软骨。与衰老和变性，所述IVD部件结构上发生变化，在组成上，并机械4。这些变化包括在NP蛋白聚糖和水合的损失，椎间盘高度降低，恶化机械能力5。这些改变是常伴促进炎症反应，以及嗜中性粒细胞和背根神经节侵入到关节间隙中，导致LBP症状6事件的级联最终细胞因子。
研究IVD变性的机制是在人类中具有挑战性，因为它往往是不可能的腰背痛发生前到变性的原因隔离。因此，简化了实验系统下到IVD器官的还原方法允许原因变量的机械微调和检查其下游效应5。该系统被减少到只有天然细胞群和周围的细胞外基质，从而使的外部刺激对IVD退化的影响的直接解释。此外，更低的成本和小鼠模型的可扩展性，以及大量的转基因动物7，允许吨体外诊断退行性机制和潜在的治疗，他迅速有针对性的筛选。在这里，我们描述了其中IVD细胞和组织稳定性被保持超过21天，给予体外诊断稳态，机械，结构，和炎性样式向特定焦点鼠器官培养系统。使用这种方法，我们重点监测的体外诊断试剂在刺致损伤模型8功能的改变，了解背后的椎间盘退变的机制。此外，我们描述这个器官培养方法的几个应用程序，以进行进一步的研究，包括对比增强显微CT成像和IVD的三维高分辨率模型。
Protocol
所有的动物实验均按照圣路易斯动物研究委员会的华盛顿大学进行。 1.动物获得的小鼠的两个菌株：10周龄的BALB / C（N = 6，BALB-M，BALB / cAnNTac）和10周龄核因子kappa-B-萤光素酶报道动物（NF-κβ-LUC）饲养在的BALB / c背景（N = 6，BALB / C-TG（RELA-LUC）31Xen）。 之前夹层，在2.5-3升/分钟的流速进行5分钟，然后停留时间的另外的2分钟安乐死与CO 2过量的动物。 <…
Representative Results
图2-3示出的蛋白聚糖分布，NF-κB表达，刚度，粘度，椎间盘高度，和湿重为培养的小鼠体外诊断的代表性结果。如果适当地培养，对照组的IVD参数不应该从新鲜组显著不同。如果培养物感染或以其它方式受损，对照组将从新鲜组不同，特别是在NF-κB表达和蛋白聚糖分布（未示出结果）。 图器官培养的使用对比增强显微CT获得IVD的三维模型4-5示出的应用程序;?…
Discussion
该协议概述了监测在IVD的生物变化鼠FSU的重点器官培养。这些文化的成功维护需要仔细无菌操作技术。特别地，解剖步骤2.1-2.6和培养步骤3.1-3.6需要特别小心，以确保保持在无菌条件，并且这些步骤应当在分离的过程室具有HEPA气流以减少污染物优选进行。由于解剖过程导致的创伤组织中，在3.5的24小时的预处理周期是必需的所有处理组，包括对照。生存力测定法可以用于?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由华盛顿大学骨骼肌肉研究中心（NIH P30 AR057235），分子影像中心（NIH P50 CA094056），力学生物学培训资助（NIH 5T32EB018266），美国国立卫生研究院R21AR069804和NIH K01AR069116支持。笔者想感谢帕特里克·黄为他的数据收集捐款。
Materials
| 96 well plate | Midwest Scientific | TP92096 | Used for biochemical assays |
| 24 well plate | Midwest Scientific | TP92024 | Used for organ culture |
| 25 ml pipettes | Midwest Scientific | TP94024 | Used for organ culture |
| 10 ml pipettes | Midwest Scientific | TP94010 | Used for organ culture |
| 5 ml pipettes | Midwest Scientific | TP94005 | Used for organ culture |
| 500 ml bottle top filters, 22um | Midwest Scientific | TP99505 | Used for filter media |
| 10 ul pipette tips | Midwest Scientific | NP89140098 | Used for biochemical assays |
| 200 ul pipette tips | Midwest Scientific | NP89140900 | Used for biochemical assays |
| 1000 ul pipette tips | Midwest Scientific | NP89140920 | Used for biochemical assays |
| DMEM /F-12 | Invitrogen | 11330032 | Used for culture media |
| Optiray 350 | Guebert | 19133341 | Used for contrast enhanced microCT |
| Fetal Bovine Serum | Sigma | F2442 | Used for culture media |
| Penicillin Streptomycin | Sigma | P4333 | Used for culture media |
| Tetrazolium Blue Chloride | Sigma | T4375 | Used for biochemical assays |
| D-Luciferin | Sigma | L6152 | Used for bioluminescence imaging |
| Chondroitin Sulfate | Sigma | C9819 | Used for biochemical assays |
| 10% Phosphomolybdic Acid Solution | Sigma | HT152 | Used for contrast enhanced microCT |
| Safranin O | Sigma | S8884 | diluted to .1% concentration (in water) |
| Fast Green FCF | Sigma | F7258 | .001% concentration |
| Papain from papaya latex | Sigma | P3125 | Used for biochemical assays |
| DAPI | Sigma-Aldrich | D9542 | Nucleic acid staining |
| Cyanoacrylate Glue | Loctite | 234790 | Adhesive |
| 1.5 ml Microcentrifuge Tubes | Fischer Scientific | S348903 | Used for biochemical assays |
| Big Equipment | |||
| BioDent | ActiveLife | For mechanical testing | |
| Cytation 5 | Biotek | Spectrophotometer | |
| AxioCam503 | Carl Zeiss AG | Microscope | |
| VivaCT40 | Scanco | MicroCT | |
| Analytical balance | Denver Instrument Company | A-200DS | Analytical balance |
| Incubator HERAcell 150i | Thermo Scientific | Organ Culture | |
| Dissection Scope | VistaVision | Used during dissection | |
| Laser Micrometer | Keyence | LK-081 | Measuring disc height |
| Microcentrifuge 5810 R | Eppendorf | Used for biochemical assays | |
| Microtome | Leica | RM2255 | Used for histology |
| Software | |||
| Prism 7 | GraphPad | For statistics | |
| MATLAB R2014a | Mathworks | For modeling | |
| Osiri-LXIV | Pixmeo | Open Source | |
| MeshLab v1.3.3 | Visual Computing Lab – ISTI – CNR | Open Source | |
| PreView/FEBio 2.3 | Utah MRL & Columbia MBL | Open Source | |
| ImageJ | NIH | ||
| Microsoft Excel | Windows | ||
| Dissection Tools | |||
| Cohan-Vannas Spring Scissors | Fine Science Tools | 15000-02 | Or any nice pair of spring scissors |
| Fine Scissors – Sharp (small) | Fine Science Tools | 14060-09 | |
| Fine Scissors – Sharp (larger) | Fine Science Tools | 14060-11 | |
| Dumont #5 Forceps | Fine Science Tools | 11252-40 | At least 2; can also use #3 |
| Extra Fine Graefe Forceps, serrated | Fine Science Tools | 11150-10 | At least 2 |
| Micro-Adson Forceps, serrated | World Precision Instruments | 503719-12 | |
| Micro-Adson Forceps, teeth | World Precision Instruments | 501244 | |
| Scalpel Handle – #3 | Fine Science Tools | 10003-12 | |
| Scalpel Handle – #4 | Fine Science Tools | 10004-13 | |
| Scalpel Blades – #23 | Fine Science Tools | 10023-00 | |
| Insect Pins , size 000 | Fine Science Tools | 26000-25 | |
| 27G Needle | BD PrecisionGlide Needles | BD305109 |
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Cite This Article
Liu, J. W., Lin, K. H., Weber, C., Bhalla, S., Kelso, S., Wang, K., Tang, S. Y. An In Vitro Organ Culture Model of the Murine Intervertebral Disc. J. Vis. Exp. (122), e55437, doi:10.3791/55437 (2017).