编制完整的牛尾巴器官培养的椎间盘
Summary
该协议说明了牛尾骨器官培养的椎间盘的收获技术<em>在体外</em>机关文化。
Abstract
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。
Protocol
1。椎间盘的收获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。 准备大砧板，砧板的上方（图2）无菌工作站和仪器准备。 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。 准备一个盆，并用自来水稀释1:100的优碘的解决方案。 沉浸在整个流域包含1％的优碘溶液5分?…
Discussion
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，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这个项目是由瑞士国家科学基金会（SNF＃310030-127586/1）的支持。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Fresh bovine intervertebral disc tissue from bovine tails, from local slaughter house (ideally within hours post-mortem and without skin). | |||
| Pulsavac Plus AC System | Zimmer inc., Switzerland | 00-5150-486-01 | Best performance with the hip-spray head and with AC power supply (the one with the 8 AA battery pack does also work but is less convenient) |
| High Capacity Fan Spray w/Splash Shield, 12.7cm length | Zimmer inc., Switzerland | 00-5150-175-00 | There are several spray heads available, we tested this one successfully |
| Scalpel blades #22 and #10 | Swann-Morton, England | #10: 0201 #22: 0208 |
|
| Scalpel blade holder # 3 and #4 | Hausmann, Germany | #3: 06.103.00 #4: 06.104.00 |
|
| Lutz industrial blade | Lutz, Germany | 1022.0884 | |
| Phosphate buffered Saline (PBS) | Invitrogen, Switzerland | 10010-023 | |
| Dulbecco’s Modified Eagle Medium (DMEM) | Gibco, Switzerland | 11960-044 | |
| Lactated Ringer’s solution (without glucose) | Bichsel, Switzerland | 133 0002 | |
| 6-well multi-well plate | TPP, Switzerland | 92006 | |
| Betadine solution | Mundipharma, Switzerland | 10055025 | |
| Surgical skin marker | Porex Surgical, Switzerland | 9560 | |
| Large cutting board | Any brand is possible | ||
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Tags
Intact Bovine Tail Intervertebral DiscsOrgan CultureIntervertebral Disc (IVD)SpineNucleus Pulposus (NP)Annulus Fibrosus (AF)Cartilaginous EndplatesDegenerative Intervertebral Disc Disease (DDD)Disc HerniationsDiscogenic PainIn Vitro Organ CultureLive Disc CellsMechano-biological AspectsBovine Coccygeal IVDsHuman Lumbar IVDsCell DensityCell PopulationDimensionsBovine Caudal IVD Harvesting TechniquesCartilaginous EndplatesBony EndplatesAvascular OrgansDiffusionCapillary Buds
Cite This Article
Chan, S. C., Gantenbein-Ritter, B. Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture. J. Vis. Exp. (60), e3490, doi:10.3791/3490 (2012).