这个项目是由瑞士国家科学基金会（SNF＃310030-127586/1）的支持。

1。椎间盘的收获<ol><li>全长牛尾巴是从本地屠宰场获得，没有皮肤，因为皮肤的存在增加了受污染的机会（图2）如果可能的话。 </li><li>准备大砧板，砧板的上方（图2）无菌工作站和仪器准备。 </li><li>准备下的无菌层流罩无菌纱布蘸0.9％氯化钠氯化包含55mm的柠檬酸钠和投入以及每6孔板。 </li><li>准备一个盆，并用自来水稀释1:100的优碘的解决方案。 </li><li>沉浸在整个流域包含1％的优碘溶液5分钟的牛尾巴。 </li><li>简要干尾，用无菌纱布放在无菌工作站。 </li><li>小心地取出22＃手术刀刀片肌肉周围的尾巴。 </li><li>印章远离尾巴的部分是没有必要，通常两端的尾巴，其中包含比较大和非常小体外诊断（图3）。 </li><li>修剪进一步的肌肉和肌腱周围的体外诊断用手术刀刀片＃10。小心不要切光盘的外圆。 </li><li>标记前手术皮肤标记的体外诊断试剂（这一步是可选的，它有助于确定我们的生物反应器的轴向旋转中心）。 </li><li>找到移动的尾巴轻轻的体外诊断试剂和脊椎连接点。感觉沉闷的手术刀刀片IVD和骨之间的边界。裂解网站应该对脊椎的体外诊断试剂1-2毫米。广场定制的工业刀片座，切割现场（图4）。 </li><li>顺劈斩，体外诊断试剂和脊椎定制的工业刀片座（图3）上敲敲打打。 </li><li>重复的体外诊断椎体连接的另一端。 </li><li>裹孤立的体外诊断用无菌纱布蘸含55mm的柠檬酸钠0.9％氯化钠氯化。 </li><li>所需的样本数（通常大约6可以继续隔离的体外诊断试剂直径10-20毫米之间，收获）。 </li><li>喷射灌洗系统（吉玛，INC。）连接无菌乳酸林格氏液，5L包装袋是一个有用的的大小（或者无菌PBS或0.9％生理盐水溶液）。 </li><li>保持与镊子和喷射灌洗既终板表面的两侧，使用吉玛Pulsavac喷射灌洗系统（图1C）的体外诊断试剂。喷枪应终板表面30之间的角度拍摄 - 60 °左右，每边30年代。 （图1和7） </li><li>把体外诊断试剂的6孔培养板，敷以浸湿纱布，而射流清洗另一张光盘样本。 </li><li>体外诊断是器官培养和下游的应用程序（例如，细胞活力，机械负荷，机关文化）（图1d）。 </li></ol> 2。代表性的成果成果测量，以判断一个成功的椎间盘制剂可扩散实验，其中一个小分子量的荧光染料（如procion红）准备为1％溶液在PBS洗涤2。体外诊断试剂是保持至少100毫升染料溶液和染料，然后允许在自由膨胀扩散到24小时的体外诊断试剂的被动。体外诊断试剂是闪存液氮冷冻，并带回至室温的丙酮转移系列（第一预冷在-80 ° C，然后-20 ° C，然后4 ° C和最后到室温脱水）。光盘可以被嵌入在聚甲基丙烯酸甲酯（PMMA）和切用锋利的刀片生产100微米厚的部分。然后，这些部分可以被视为由任何标准来衡量明显微镜，但优先使用荧光显微镜，因为procion红色会发出红色荧光（图5-6）。 trabeculi骨表面看起来很干净后喷射lavaging步骤（图7）。 一个成功的器官培养的参数是所有没有污染，保持细胞活力（图8-9），椎间盘高度的第一和整体的“光盘的完整性”，与组织学切片和番红O /快绿染色或迈耶的Hematoxilin 3测定。我们开发的协议和宏别处发表 4,5染色现场椎间盘组织，并在三维扫描单元使用共聚焦激光扫描显微镜和活/死染色试剂盒（分子探针，Invitrogen公司，巴塞尔，瑞士）的可行性。我们进一步发展的宏和程序执行自动化的细胞计数，估计在三维组织或3D运营商使用美国国立卫生研究院ImageJ平台4-6细胞活力。已提出替代的协议，以确定由最初的使用顺序使用链霉蛋白酶和胶原酶类型的细胞外基质的消化消化细胞椎间盘细胞的可行性2，然后染色的细胞悬液7。一个早期版本的协议涉及孵育10min后用含光盘收获前器官立方米十倍较为集中的青霉素/链霉素浓度一步lture。与Jet灌洗一步一步变得过时，因为这一步，似乎有利于培养目的。 <img alt="图1" src="/files/ftp_upload/3490/3490fig1.jpg" /> 图1。概述的方法步骤，准备完整的终板和骨薄薄的一层（1 - 2MM）牛尾骨椎间盘。一）椎间盘（IVD）和它的营养通路的示意图。二）程序与一个定制的刀片持有者和工业锋利的刀片切出的体外诊断试剂。 C）喷射灌洗步骤，以确保高的细胞活力和保持终板和附骨双方1-2毫米，使养分交换。 d）最后的体外诊断试剂，可以培养生物反应器的样品室或用于任何下游应用。 <img alt="图2" src="/files/ftp_upload/3490/3490fig2.jpg" /> 图2上：全长新鲜牛尾巴（理想obta独立非执行董事内2-3小时验尸报告，以确保高细胞活力。底部：X射线图像，说明存在着生长板（GP），二次骨化中心之间的骨性终板（EP）和脊椎机构（VB），约6个月年龄的牛尾巴。 <img alt="图3" src="/files/ftp_upload/3490/3490fig3.jpg" /> 图3 A）工具，在无菌条件下解剖牛尾巴。二）定制的刀片人切出动感段从牛尾巴在行动的同时，减少椎间盘。 c）边的一个工业标准刀片（Lutz称，德国）的定制刀片人的观点。 <img alt="图4" src="/files/ftp_upload/3490/3490fig4.jpg" /> 图4。绘图和图片的牛尾巴取出后，周围的肌肉和韧带，切出动感的细分说明裂解网站。部分椎骨应“破解”无线网络TH定制的刀片持有人远离软骨终板的理想1-2毫米，以确保充分的骨面。 <img alt="图5" src="/files/ftp_upload/3490/3490fig5.jpg" /> 图5（A）和（二）有效的清洁喷射灌洗步骤示范前。请注意干净椎间盘段骨表面喷涂程序后。 <img alt="图6" src="/files/ftp_upload/3490/3490fig6.jpg" /> 图6。自由膨胀扩散新鲜配制切除牛尾巴椎1％procion红色溶液24小时留在光盘（体外诊断）的实验，证明生长板堵塞的效果。一个数字化X射线的矢状查看体外诊断仍然包含通过尾骨牛椎间盘植体与软​​骨和骨性终板的生长板和矢状切骨荧光显微镜图像的第二层后24H自由扩散。大奖赛：生长板，2ndry二氧化碳：二次骨化中心。 <img alt="图7" src="/files/ftp_upload/3490/3490fig7.jpg" /> 图7。自由膨胀实验准备新鲜的牛切除尾巴椎为1％procion红色溶液中24小时左光盘（体外诊断），展示清洁灌洗治疗效果的喷气。矢状厚〜1.5 mmthick骨端板，喷气灌洗治疗（左）控制端接受未经处理（右）喷射灌洗治疗切除的牛体外诊断（〜100毫米）。入口显示齐默尔伤口清创系统中使用的喷雾模式，这是24小时的牛在1％procion红色椎间盘植的自由扩散实验。 <img alt="图8" src="/files/ftp_upload/3490/3490fig8.jpg" /> 图8。bovin采取活/死的共焦成像堆栈的预测图像（250微米）发送椎间盘段准备与自由膨胀下保持为0和14天分别为（N​​P）的髓核，内层纤维环（IA）的和外层纤维环（OA）的喷射灌洗方法。 =钙黄绿素AM（乙酰甲酯）染色的活细胞，红细胞=死细胞的细胞核二聚体- 1乙锭染色绿色细胞。 <img alt="图9" src="/files/ftp_upload/3490/3490fig9.jpg" /> 图9。细胞椎光盘，用活组织和3D堆栈扫描共聚焦激光扫描显微镜的可行性。自由膨胀条件下髓核（NP）和环形纤维化（AF）细胞存活率超过21天的文化。 （平均± SEM，n = 6）统计差异进行了测试，使用非参数克鲁斯卡尔 - 沃利斯秩和检验组间签署。第21天，在NP的所有其他的时间点之间的显著差异。虽然在自动对焦显著差异之间 - 7天被发现0天。 （* P &lt;0.05，** P &lt;0.01）。

<ol>
	<li>Lee, C. R., Iatridis, J. C., Poveda, L., Alini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+organ+culture+of+the+bovine+intervertebral+disc:+effects+of+vertebral+endplate+and+potential+for+mechanobiology+studies.">In vitro organ culture of the bovine intervertebral disc: effects of vertebral endplate and potential for mechanobiology studies.</a> Spine (Phila Pa 1976). 31, 515-522 (1976).</li><li>Chan, S. C. W., Gantenbein-Ritter, B., Leung, V. Y., Chan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreserved+intervertebral+disc+with+injected+bone+marrow-derived+stromal+cells:+a+feasibility+study+using+organ+culture.">Cryopreserved intervertebral disc with injected bone marrow-derived stromal cells: a feasibility study using organ culture.</a> Spine. J. 10 (6), 486-496 (2010).</li><li>Chan, S. C., Ferguson, S. J., Wuertz, K., Gantenbein-Ritter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Response+of+the+Intervertebral+Disc+to+Repetitive+Short+Term+Cyclic+Torsion.">Biological Response of the Intervertebral Disc to Repetitive Short Term Cyclic Torsion.</a> Spine (Phila Pa 1976). , (2011).</li><li>Gantenbein-Ritter, B., Sprecher, C. M., Chan, S., Illien-J&#252;nger, S., Grad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+imaging+protocols+for+live/dead+staining+in+three-dimensional+carriers.">Confocal imaging protocols for live/dead staining in three-dimensional carriers.</a> Methods Mol. Biol. 740, 127-140 (2011).</li><li>Gantenbein-Ritter, B., Potier, E., Zeiter, S., van der Werf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+three+techniques+to+determine+cell+viability+in+3D+tissues+or+scaffolds.">Accuracy of three techniques to determine cell viability in 3D tissues or scaffolds.</a> Tissue Engineering Part C Methods. 14, 353-358 (2008).</li><li>Rasband, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ImageJ. , (1997).</li><li>Haschtmann, D., Stoyanov, J. V., Ettinger, L., Nolte, L. P., Ferguson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+novel+intervertebral+disc/endplate+culture+model:+analysis+of+an+ex+vivo+in+vitro+whole-organ+rabbit+culture+system.">Establishment of a novel intervertebral disc/endplate culture model: analysis of an ex vivo in vitro whole-organ rabbit culture system.</a> Spine. 31, 2918-2925 (2006).</li><li>Gantenbein, B., Gr&#252;nhagen, T., Lee, C. R., van Donkelaar, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+organ+culturing+system+for+intervertebral+disc+explants+with+vertebral+endplates:+a+feasibility+study+with+ovine+caudal+discs.">An in vitro organ culturing system for intervertebral disc explants with vertebral endplates: a feasibility study with ovine caudal discs.</a> Spine. 31, 2665-2673 (2006).</li><li>Korecki, C. L., Kuo, C. K., Tuan, R. S., Iatridis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervertebral+disc+cell+response+to+dynamic+compression+is+age+and+frequency+dependent.">Intervertebral disc cell response to dynamic compression is age and frequency dependent.</a> J. Orthop. Res. 27, 800-806 (2009).</li><li>Korecki, C. L., MacLean, J. J., Iatridis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compression+effects+on+intervertebral+disc+mechanics+and+biology.">Dynamic compression effects on intervertebral disc mechanics and biology.</a> Spine (Phila Pa 1976). 33, 1403-1409 (1976).</li><li>Jim, B., Steffen, T., Moir, J., Roughley, P., Haglund, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+intact+intervertebral+disc+organ+culture+system+in+which+degeneration+can+be+induced+as+a+prelude+to+studying+repair+potential.">Development of an intact intervertebral disc organ culture system in which degeneration can be induced as a prelude to studying repair potential.</a> European Spine Journal. , 1-11 (2011).</li></ol>

我们什么都没有透露。

器官培养成功的第一步是要确保，不应被污染的外植体。你的程序开始之前，尾巴应该是剥皮。任何动物毛带进无菌实验室污染方面可能会有问题。牛尾巴最好应尽可能新鲜的（这会影响初始细胞活力）。此外，优碘清洗步骤是建议进一步降低污染的风险。而不是使用一个特制的刀座和锤子分开椎体的体外诊断试剂，这可能是通过组织学带锯（金刚石锯片应与PBS或林格氏液冷却切割时）。当心，?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td colspan="4">新鲜牛从牛尾巴的椎间盘组织，从本地屠宰场（理想范围内小时验尸报告和不带皮）。 </td></tr><tr><td> Pulsavac加交流系统</td><td>吉玛公司，瑞士</td><td> 00-5150-486-01 </td><td>与髋喷头和交流电源的最佳性能（与8节AA电池组一个也没有工作，但不太方便） </td></tr><tr><td>高容量喷雾风扇W /防溅挡板，12.7厘米长度</td><td>吉玛公司，瑞士</td><td> 00-5150-175-00 </td><td>有几个可用的喷头，我们测试的这一个成功</td></tr><tr><td> ＃22和＃10手术刀片</td><td>斯旺 - 莫特英格兰</td><td> ＃10：0201 ＃22：0208 </td><td></td></tr><tr><td>手术刀刀片持有人＃3和＃4 </td><td>德国郝睿强</td><td> ＃3：06.103.00 ＃4：06.104.00 </td><td></td></tr><tr><td>鲁茨工业刀片</td><td>德国卢茨</td><td> 1022.0884 </td><td></td></tr><tr><td>磷酸盐缓冲液（PBS） </td><td> Invitrogen公司，瑞士</td><td> 10010-023 </td><td></td></tr><tr><td>贝科的改良Eagle培养基（DMEM） </td><td> Gibco公司，瑞士</td><td> 11960-044 </td><td></td></tr><tr><td>乳酸林格氏液（不含葡萄糖） </td><td> Bichsel，瑞士</td><td> 133 0002 </td><td></td></tr><tr><td> 6孔多孔板</td><td>瑞士TPP， </td><td> 92006 </td><td></td></tr><tr><td>优碘的解决方案</td><td>瑞士Mundipharma， </tD&gt; <td> 10055025 </td><td></td></tr><tr><td>手术皮肤标记</td><td> Pore​​x手术，瑞士</td><td> 9560 </td><td></td></tr><tr><td>大菜板</td><td></td><td></td><td>任何品牌是可能的</td></tr></tbody></table>

preparation of intact bovine tail intervertebral discs for organ culture

椎间盘（IVD）是连接脊椎椎脊柱关节。它的功能发送加载的脊椎和脊柱的灵活性。这三个车厢组成：最内层的髓核（NP）的纤维环（AF）和两个连接双方的NP和AF椎体软骨终板无所不包。退行性椎间盘疾病（DDD）和椎间盘突出引起的椎间盘源性疼痛，可能已被确定为在现代社会的一个主要问题。为了研究体外诊断试剂变性的可能机制，在机关与现场椎间盘细胞体外培养系统具有高度吸引力。 在完整的牛尾骨体外诊断试剂的体外培养，拥有先进的有关模型的系统，它允许的生物机械方面，在控制良好的生理和机械环境的研究。牛尾巴体外诊断可以比较容易取得较高的数字D是非常相似的细胞密度，细胞人口和尺寸方面与人类的腰椎体外诊断。然而，以前的牛尾椎的IVD收获技术，保留软骨终板和骨性终板失败后1-2天的文化，因为营养途径明显血块阻塞。体外诊断是最大的股骨头缺血性机关，因此，在NP细胞的营养物质是通过从相邻椎体的毛细血管芽完全依赖扩散。终板表面的骨碎片和血块的存在可以阻碍养分扩散到光盘和妥协的细胞活力的中心。我们集团成立了一个相对快速的协议“破解”出从尾部的体外诊断为污染低风险。我们能够通过外科手术的喷气灌洗系统，从而消除血凝块和切割碎片和非常有效地重开的营养扩散途径通透的新鲜切骨性终板表面体外诊断中心。要避免双方椎体骨生长板的存在和文化之前被删除。在这段视频中，我们概述了在制备过程中的关键步骤，并展示了一个成功的器官培养，保持14天免费肿胀文化下的细胞存活率高的关键。时，所使用的机械负荷生物反应器可以保持适当的机械环境的，可以延长培养时间。这里展示的技术可以扩展到其他动物，如猪，羊和leporine尾鳍和腰椎IVD隔离。

Watch this Scientific Journal Video about Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture at JoVE.com

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

该协议说明了牛尾骨器官培养的椎间盘的收获技术<em&gt;在体外</em&gt;机关文化。

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  ...

preparation-intact-bovine-tail-intervertebral-discs-for-organ

Research

JoVE Journal

Bioengineering

14.4K 次观看.  University of Bern. 该协议说明了牛尾骨器官培养的椎间盘的收获技术在体外机关文化。

视频: Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

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 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

制备纤维蛋白三维支架干细胞培养应用

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

样品制备策略的3D细胞培养模型质谱成像

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

研究表皮生长因子受体的完整细胞使用相关显微镜的化学计量

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

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 ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

用集成电极直接量化 Transendothelial 电阻的 Organ-on-chip 系统的制作与验证

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the fragility of bone, making it fracture-prone. Owing to the large socio-economic impact of bone fracture in our society, there is a need for novel ways to assess the mechanical performance of each hierarchical level of bone. Although stiffness and strength can be probed at all scales &#8211; nano-, micro-, meso-, and macroscopic &#8211; fracture assessment has so far been confined to macroscopic testing. This limitation restricts our understanding of bone fracture and constrains the scope of laboratory and clinical studies. In this research, we investigate the fracture resistance of bone from the microscopic to the mesoscopic length scales using micro scratch tests combined with nonlinear fracture mechanics. The tests are performed in the short longitudinal orientation on bovine cortical bone specimens. A meticulous experimental protocol is developed and a large number (102) of tests are conducted to assess the fracture toughness of cortical bone specimens while accounting for the heterogeneity associated with bone microstructure.

This study assesses the fracture toughness of bovine cortical bone at the sub-meso levels using microscopic scratch tests. This is an original, objective, rigorous, and reproducible method proposed to probe fracture toughness below the macroscopic scale. Potential applications are studying changes in bone fragility due to diseases like osteoporosis.

牛皮质骨脆性评估的划痕试验

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

可伸缩、双通道、微流控芯片制造

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

跟踪开放全球伤害和治疗性能的前段器官文化平台

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

在开顶器官芯片上重建人上皮组织的细胞结构和功能

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...