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。

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<ol><li>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）。 </li><li>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。 </li><li>通过在70％乙醇浴洗澡它解剖前2分钟消毒动物的外侧区域。 </li></ol> 2.解剖<ol><li>使用小清扫剪刀，以暴露所述体腔的小鼠的背部表面上的纵向垂直切割。 </li><li>就从开始的动物的脊椎两侧有两条纵向垂直切割所述第一腰椎（L1）到尾椎（C8）。 </li><li>使用手术刀，细镊子和精细解剖剪刀，小心地从动物的体腔移除L1-C8脊椎。 </li><li>小心地移除脊柱过量软组织，同时确保不刮擦或损伤的腹侧的IVD。 </li><li>进一步解剖脊柱到在L1 / L2，L3 / L4，L5 / L6，C3 / C4，C5 / C6和C7 / C8光盘椎骨盘-椎骨脊柱功能装置（FSU）。 </li><li>冲洗FSU的在补充有1％ - 链霉素青霉素2分钟Hank平衡盐溶液中。 </li><li>随机指定的FSU的分成三组:未培养的和未处理的FSU的（新鲜），培养的但未治疗FSU的（对照），并培养和刺处理FSU的（刺）。 </li><li>卡扣冻结在-20℃的冰箱中解剖和存储后立即新鲜FSU样品用液氮。 </li></ol> 3.器官培养条件<ol><li>制备500毫升的无菌培养介质的1:1的Dulbecco改良的Eagle培养基:营养混合物F12（DMEM:F12），补充有20％胎牛血清（FBS）和1％青霉素 - 链霉素。 </li><li>用10个毫升血清移液管，移液管2毫升培养基在24孔培养板的每个孔中。 </li><li>继清扫和冲洗，将每个FSU到个体以及在填充介质的24孔板。 </li><li>孵育在维持37℃，5％CO 2，20％O 2和&gt; 90％的湿度的无菌恒温箱中的样本。 </li><li> 24个小时的初始培养期后，通过机械地使用无菌27号针头， 如图1A中的纤维环的针穿刺伤害刺组的样品。 </li><li>通过准备新的填充介质的24孔培养板更换介质每48个小时，并转移样品从旧板到新的使用无菌镊子。吸出旧媒体和dispos在生物危险废物箱旧文化板电子。 </li><li>在培养的最后一天，在孵化介质含有0.75毫克/毫升硝基蓝四唑氯化物24小时的所有样本。 </li><li>之后，冻结的所有FSU的用液氮并将其存放在-20℃冰箱中，直到准备用于机械试验或组织学（ 图1B）。 </li></ol> 4.纵向测量NF-κB <ol><li>图像的FSU的从NF-κβ萤光素酶在动物1，5，13，和19天评估NF-κB表达。 </li><li>在37℃下加入1毫克/毫升荧光素溶液10μL至每个孔，并孵育在无菌恒温箱中10分钟。 </li><li>图像使用成像系统，具有1分钟的曝光时间和第2箱设置用于生物发光的样品。 </li><li>同时，使用本机拍摄照片，并用生物发光图像覆盖它，以确定在IVD发光的解剖位置。 </li></ol><p类= "jove_title"&gt; 5。机械评估<ol><li>通过使用位移控制动态压缩9确定体外诊断的机械性能。 </li><li>使用解剖显微镜，解剖刀，和镊子，同时保持软骨终板完好并连接到IVD从每个样品取出FSU的骨椎体。 </li><li>附加的分离的体外诊断用氰基丙烯酸酯胶1厘米×1cm的×0.2cm的铝板。 </li><li>通过采取三次测量的平均使用激光千分尺每个盘的纵向轴线测量的盘的高度和宽度。由最大的光盘宽度除以平均盘高度计算椎间盘高度比率。 </li><li>使用所测量的盘高度来计算机械测试中使用的输入应变值。另外，平均盘高度约为690微米±39微米。 </li><li>将磷酸缓冲盐水浴的样品compressi下上机和预加载光盘0.02 N. </li><li>在1Hz循环压缩使用正弦波形的光盘为在1％的应变水平20个循环和5％的应变水平为3次试验的每个并记录IVD的负载和位移值。允许试验之间休息时间用于盘放松和防止损伤的10分钟。 </li><li>计算从上次周期的加载阶段的平均硬度，并计算从负载和位移数据之间的相位角的损耗角正切。 </li></ol> 6.蛋白聚糖和胶原蛋白的定量<ol><li>以下机械测试，通过将所述分离的IVD在预配衡的离心管中，并用分析天平测量重量测量盘湿重。 </li><li>消化分离的IVD在250μl木瓜蛋白酶消化溶液（0.1M醋酸钠，0.01M EDTA，0.005M的盐酸半胱氨酸，pH值6.4），使用块加热器在65℃下过夜。 </li><li> 10分钟离心分离的样品在2000 XG并收集上清液。 </li><li>通过使用二甲基亚甲蓝（DMMB）与硫酸软骨素标准测定法测量的IVD的蛋白多糖含量。 </li><li>制备DMMB溶液（21毫克DMMB，5毫升无水乙醇中，在1升的DDH 2 O2克甲酸钠）。 </li><li>移液管的标准30μL和样品中，在96孔板中一式三份，用在每个DMMB溶液250μL以及沿。 </li><li>读板的吸光度在用分光光度计525纳米。 </li><li>测量使用根据制造商的说明书一羟脯氨酸测定试剂盒的胶原含量。 </li></ol> 7.组织学<ol><li>固定的样品在4％多聚甲醛的子集24个小时，在5％甲酸去钙化48个小时，用乙醇脱水，并在石蜡中嵌入。 </li><li>使用切片机，部分样品在矢状位10米微米厚，并应用部分，以载玻片上。染色使用番红O /快Gr而部分EEN和DAPI。 </li><li>使用光学显微镜，图像滑动在10X放大倍数。 </li></ol> 8.对比增强microComputed断层摄影术（微CT） <ol><li>在使用纵向碘佛醇对比度增强10，和终端磷钼酸（PMA）对比度增强在7天时间点的0，2,5，和7天的时间点扫描样品的一个子集。 </li><li>之前微CT扫描时间4个小时，在37℃下添加350毫克/毫升碘佛醇溶液到培养基为大约50毫克/毫升含碘碘佛醇溶液的最终浓度300μL，并培育在无菌恒温箱中4 H。 </li><li>温育后，包装在1毫升微量离心管中在无菌纱布和代替样品。 </li><li>在微CT系统的地方样品和扫描在45 keVp，177μA，10.5微米体素尺寸，和300毫秒的整合。 </li><li>从微型电脑为DICOM文件导出数据和使用软件的可视化（ <em&gt;例如，OsiriX）。 </li><li>在7天的时间点，固定使用24小时4％多聚甲醛溶液，接着用5％PMA溶液3天的样品，和扫描使用相同的设置与在步骤8.4中使用。 </li></ol> 9.三维有限元建模<ol><li>段使用的软件有手动阈值（ 例如 ，OsiriX）的体外诊断试剂的DICOM文件。 </li><li>转换IVD的NP和AF卷成使用Meshlab基于体素的有限元网格。 </li><li>结合NP和AF的微结构，以形成一个完整的IVD​​和在有限元软件应用通过实验确定的边界条件。 </li></ol>

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这个手稿的作者宣称，他们没有竞争的经济利益。

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?...

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。

<table><tbody><tr><td>96 well plate</td><td>Midwest Scientific</td><td>TP92096</td><td>Used for biochemical assays</td></tr><tr><td>24 well plate</td><td>Midwest Scientific</td><td>TP92024</td><td>Used for organ culture</td></tr><tr><td>25 mL pipettes</td><td>Midwest Scientific</td><td>TP94024</td><td>Used for organ culture</td></tr><tr><td>10 mL pipettes</td><td>Midwest Scientific</td><td>TP94010</td><td>Used for organ culture</td></tr><tr><td>5 mL pipettes</td><td>Midwest Scientific</td><td>TP94005</td><td>Used for organ culture</td></tr><tr><td>500 mL bottle top filters, 22 &amp;micro;m</td><td>Midwest Scientific</td><td>TP99505</td><td>Used for filter media</td></tr><tr><td>10 &amp;micro;L pipette tips</td><td>Midwest Scientific</td><td>NP89140098</td><td>Used for biochemical assays</td></tr><tr><td>200 &amp;micro;L pipette tips</td><td>Midwest Scientific</td><td>NP89140900</td><td>Used for biochemical assays</td></tr><tr><td>1,000 &amp;micro;L pipette tips</td><td>Midwest Scientific</td><td>NP89140920</td><td>Used for biochemical assays</td></tr><tr><td>DMEM/F-12</td><td>Invitrogen</td><td>11330032</td><td>Used for culture media</td></tr><tr><td>Optiray 350</td><td>Guebert</td><td>19133341</td><td>Used for contrast enhanced microCT</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F2442</td><td>Used for culture media</td></tr><tr><td>Penicillin Streptomycin&amp;nbsp;</td><td>Sigma</td><td>P4333</td><td>Used for culture media</td></tr><tr><td>Tetrazolium Blue Chloride</td><td>Sigma</td><td>T4375</td><td>Used for biochemical assays</td></tr><tr><td>D-Luciferin</td><td>Sigma</td><td>L6152</td><td>Used for bioluminescence imaging</td></tr><tr><td>Chondroitin Sulfate</td><td>Sigma</td><td>C9819</td><td>Used for biochemical assays</td></tr><tr><td>10% Phosphomolybdic Acid Solution</td><td>Sigma</td><td>HT152</td><td>Used for contrast enhanced microCT</td></tr><tr><td>Safranin O</td><td>Sigma</td><td>S8884</td><td>diluted to .1% concentration (in water)</td></tr><tr><td>Fast Green FCF</td><td>Sigma</td><td>F7258</td><td>.001% concentration</td></tr><tr><td>Papain from papaya latex</td><td>Sigma&amp;nbsp;</td><td>P3125</td><td>Used for biochemical assays</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td>Nucleic acid staining</td></tr><tr><td>Cyanoacrylate Glue</td><td>Loctite</td><td>234790</td><td>Adhesive&amp;nbsp;</td></tr><tr><td>1.5 mL Microcentrifuge Tubes</td><td>Fischer Scientific</td><td>S348903</td><td>Used for biochemical assays</td></tr><tr><td>&lt;strong&gt;Big Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BioDent</td><td>ActiveLife</td><td></td><td>For mechanical testing</td></tr><tr><td>Cytation 5</td><td>Biotek</td><td></td><td>Spectrophotometer</td></tr><tr><td>AxioCam503</td><td>Carl Zeiss AG</td><td></td><td>Microscope</td></tr><tr><td>VivaCT40</td><td>Scanco</td><td></td><td>MicroCT</td></tr><tr><td>Analytical balance</td><td>Denver Instrument Company</td><td>A-200DS</td><td>Analytical balance</td></tr><tr><td>Incubator HERAcell 150i</td><td>Thermo Scientific</td><td></td><td>Organ Culture</td></tr><tr><td>Dissection Scope</td><td>VistaVision</td><td></td><td>Used during dissection</td></tr><tr><td>Laser Micrometer</td><td>Keyence</td><td>LK-081</td><td>Measuring disc height</td></tr><tr><td>Microcentrifuge 5810 R</td><td>Eppendorf</td><td></td><td>Used for biochemical assays</td></tr><tr><td>Microtome</td><td>Leica&amp;nbsp;</td><td>RM2255</td><td>Used for histology</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Prism 7</td><td>GraphPad</td><td></td><td>For statistics</td></tr><tr><td>MATLAB R2014a</td><td>Mathworks</td><td></td><td>For modeling</td></tr><tr><td>Osiri-LXIV</td><td>Pixmeo</td><td></td><td>Open Source</td></tr><tr><td>MeshLab v1.3.3</td><td>Visual Computing Lab - ISTI - CNR</td><td></td><td>Open Source</td></tr><tr><td>PreView/FEBio 2.3</td><td>Utah MRL &amp;amp; Columbia MBL</td><td></td><td>Open Source</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td></td></tr><tr><td>Microsoft Excel</td><td>Windows</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Dissection Tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cohan-Vannas Spring Scissors&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>15000-02</td><td>Or any nice pair of spring scissors</td></tr><tr><td>Fine Scissors - Sharp&amp;nbsp; (small)</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>14060-09</td><td></td></tr><tr><td>Fine Scissors - Sharp&amp;nbsp; (larger)</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>14060-11</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>11252-40</td><td>At least 2; can also use #3&amp;nbsp;</td></tr><tr><td>Extra Fine Graefe Forceps, serrated</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>11150-10</td><td>At least 2</td></tr><tr><td>Micro-Adson Forceps, serrated</td><td>World Precision Instruments</td><td>503719-12</td><td></td></tr><tr><td>Micro-Adson Forceps, teeth</td><td>World Precision Instruments</td><td>501244</td><td></td></tr><tr><td>Scalpel Handle - #3</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>10003-12</td><td></td></tr><tr><td>Scalpel Handle - #4</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>10004-13</td><td></td></tr><tr><td>Scalpel Blades - #23</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>10023-00</td><td></td></tr><tr><td>Insect Pins, size 000</td><td>Fine Science Tools</td><td>26000-25</td><td></td></tr><tr><td>27 G Needle</td><td>BD PrecisionGlide Needles</td><td>BD305109</td><td></td></tr></tbody></table>

an in vitro organ culture model of the murine intervertebral disc

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。

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;?...

Watch this Scientific Journal Video about An In Vitro Organ Culture Model of the Murine Intervertebral Disc at JoVE.com

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

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。

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

an-in-vitro-organ-culture-model-of-the-murine-intervertebral-disc

Research

JoVE Journal

Bioengineering

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

9.5K 次观看.  Washington University in St. Louis. 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。 

视频: An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

科研

生物工程

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

编制完整的牛尾巴器官培养的椎间盘

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

小鼠模型肠组织工程

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

对于中吸入有毒化学测试体外细胞培养模型

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Ovine Lumbar Intervertebral Disc Degeneration Model Utilizing a Lateral Retroperitoneal Drill Bit Injury

Intervertebral disc degeneration is a significant contributor to the development of back pain and the leading cause of disability worldwide. Numerous animal models of intervertebral disc degeneration have been developed. The ideal animal model should closely mimic the human intervertebral disc with regard to morphology, biomechanical properties and the absence of notochordal cells. The sheep lumbar intervertebral disc model fulfils these criteria. We present an ovine model of intervertebral disc degeneration utilizing a drill bit injury through a lateral retroperitoneal approach. The lateral approach significantly reduces the incision and potential morbidity associated with the traditional anterior approach to the ovine spine. Utilization of a drill-bit method of injury affords the ability to produce a consistent and reproducible injury, of precise dimensions, that initiates a consistent degree of intervertebral disc degeneration. The focal nature of the annular and nucleus pulposus defect more closely mimics the clinical condition of focal intervertebral disc herniation. Sheep recover rapidly following this procedure and are typically mobile and eating within the hour. Intervertebral disc degeneration ensues and sheep undergo necropsy and subsequent analysis at periods from eight weeks. We believe that the drill bit injury model of intervertebral disc degeneration offers advantages over more conventional annular injury models.

Intervertebral disc degeneration is a significant contributor to back pain and a leading cause of disability worldwide. Numerous animal models of intervertebral disc degeneration exist. We demonstrate an ovine model of intervertebral disc degeneration, utilizing a drill bit, which achieves a consistent disc injury and reproducible level of disc degeneration.

Ovine Lumbar椎间盘退变模型利用横向腹膜后位钻伤

Intervertebral disc degeneration is a significant contributor to the development of back pain and the leading cause of disability worldwide. Numerous ...

医学

A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.

Symptomatic intervertebral disc (IVD) degeneration (IDD) is a major socioeconomic burden and is characterized by inflammation and tissue degradation. Due to the lack of causative therapies, there is an urgent need for innovative experimental organ culture models to study the mechanisms involved in the progression of the disease, find therapeutic targets, and reduce the need for animal models. We here present a novel, three-dimensional organ culture model protocol mimicking the proinflammatory and catabolic microenvironment, which is present during IDD. 
Initially, bovine caudal IVDs were dissected, cleaned, and cultured in the tissue culture medium. Dynamic physiologic or pathologic loading was applied in a custom-made bioreactor for 2 hours per day. IVDs were assigned to a control group (high glucose medium, physiological loading, phosphate-buffered saline injection) and a pathological group (low glucose medium, pathological loading, tumor necrosis factor-alpha injection) for four days. Gene expression analysis from collected nucleus pulposus cells of the IVDs and enzyme-linked immunosorbent assay of the conditioned organ culture media was performed. 
Our data revealed a higher expression of inflammatory markers and reduced disc heights after loading in the pathological group compared to the control group. This protocol is reliable to simulate IVD inflammation and degeneration and can be further expanded to broaden its application scope.

This protocol presents a novel experimental model of proinflammatory, degenerative bovine organ culture to simulate early-stage intervertebral disc degeneration.

模拟早期椎间盘疾病的发炎性、退行性器官培养模型。

Symptomatic intervertebral disc (IVD) degeneration (IDD) is a major socioeconomic burden and is characterized by inflammation and tissue degradation. Due ...

Optical Sectioning and Visualization of the Intervertebral Disc from Embryonic Development to Degeneration

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To decode disc degeneration and to be able to develop regenerative approaches a thorough understanding of the cellular biology of the IVD is essential. One aspect of this biology that still remains unanswered is the question of how cells are spatially arranged in a physiological state and during degeneration. The biological properties of the IVD and its availability make this tissue difficult to analyze. The present study investigates spatial chondrocyte organization in the anulus fibrosus from early embryonic development to end-stage degeneration. An optical sectioning method (Apotome) is applied to perform high resolution staining analyses using bovine embryonic tissue as an animal model and human disc tissue obtained from patients undergoing spine surgery. From a very high chondrocyte density in the early embryonic bovine disc, the number of cells decreases during gestation, growth, and maturation. In human discs, an increase in cellular density accompanied the progression of tissue degeneration. As had already been demonstrated in articular cartilage, cluster formation represents a characteristic feature of advanced disc degeneration.

We present a method to investigate spatial chondrocyte organization in the anulus fibrosus of the intervertebral disc using an optical sectioning method.

椎间盘从胚胎发育到退化的光学切片和可视化

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To ...

生物学

Generating a Pro-Inflammatory Organ Culture Model to Simulate an Early Intervertebral Disc Disease

Source: Saravi, B., et al. <a href="https://app.jove.com/v/62100">A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.</a> J. Vis. Exp. (2021).
This video demonstrates a technique for culturing bovine intervertebral discs in bioreactors to develop a three-dimensional organ culture model. Specific nutrient-deprived media and degenerative pressure conditions are applied to the discs, mimicking the pro-inflammatory and degenerative microenvironment observed during early-stage intervertebral disc disease. The disc heights are measured post-treatment to analyze the effects of nutrient deprivation and degenerative loading.

生成促炎器官培养模型以模拟早期椎间盘疾病

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Dissection of the bovine ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

Generating an Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease

Source: Saravi, B. et al., <a href="https://app.jove.com/v/62100">A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.</a>&#160;J. Vis. Exp. (2021).
This video demonstrates the development of a pro-inflammatory and degenerative organ culture model for early-stage intervertebral disc (IVD) disease using TNF-&#945;. It outlines the procedure for injecting TNF-&#945; into the intervertebral disc and explains the mechanisms through which TNF-&#945; induces inflammation and degradation of the IVD.

Source: Saravi, B. et al., A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.&#160;J. Vis. Exp. ...

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