小鼠模型肠组织工程
Summary
本文及随附视频我们的协议，用于生成在小鼠小肠组织工程，使用类器官单位支架的方法。
Abstract
组织工程化小肠（TESI）已经成功地被用于拯救Lewis大鼠大量小肠切除后，在40天内恢复到术前的权重。1在人类中，大量小肠切除会导致短肠综合征，功能性吸收不良国家赋予重大的发病率，死亡率和医疗费用，包括肠外营养的依赖，肝功能衰竭和肝硬化，需要multivisceral器官移植。本文中，我们描述和记录我们的协议在小鼠模型中创建组织工程小肠一个多细胞类器官单位支架的方法。类器官单位是多细胞聚集来自肠道粘膜与间充质元素，包含3之间的关系，保留了肠道干细胞小生境。在目前和未来的研究中，我们的技术过渡到的鼠标将允许利用转基因工具，可以在这个物种在TESI形成的过程进行调查。5免疫功能低下小鼠品系的可用性也将允许我们应用该技术对人体肠道组织和优化形成的人TESI作为小鼠异种移植前的过渡到人类。我们的方法，采用良好生产规范（GMP）试剂和材料已经被批准用于人类患者，因此提供了一个重要的方法，依赖于脱细胞动物组织的优势。这种方法的最终目标是将其翻译给人类作为再生医学的短肠综合征的治疗策略。
Protocol
1。类器官单位准备仪器适用于鼠标清扫术（剪刀和镊子）应通过高压灭菌器灭菌。 人道安乐死的供体小鼠根据当地IACUC协议。确保该动物是死的，然后再继续。 车型中线切口进入腹膜腔。皮瓣可以反映需要改进的曝光。 剔骨的小肠和Treitz韧带远端分。分离小肠从肠系膜使用锋利和温和的钝性分离。确定回盲部和分裂小肠5毫米近端。 使用剪刀，打开肠道纵向沿…
Representative Results
图1示出了一个整体的协议架构的记录在这里。此协议的最终结果是一个球形或球状结构的组织工程小鼠小肠的管腔，粘膜，粘膜下层，和周围肌层。 图2A示出一个典型的地球比较起始聚合物支架。 图2B中显示的相同的构造与急剧双瓣壳，以揭示其管腔。 图3演示了一个苏木精/伊红染色的石蜡安装截面一个典型的成功构建体，4周后的潜伏期。在?…
Discussion
我们提出了一个协议，用于组织工程在小鼠小肠类器官单位支架的方法。最关键的步骤是类器官单位准备。必须小心，以充分清洗和机械处理的组织，但等于必须小心不overdigest，或overtriturate的类器官单位消化后进行（步骤1.11）。类器官单位如果做到这一点，可以减少到单细胞，这可能会丢失在步骤1.12的上清液，并不大可能生存产生组织工程小肠，作为肠干细胞小生境将不被保存在这的情况下。…
Divulgations
The authors have nothing to disclose.
Acknowledgements
特雷西C. Grikscheit，埃里克R.巴特尔，和弗雷德里克·G.萨拉所支持的加州再生医学研究所（CIRM），授权号码RN2-00946-1（TCG）和TG2-01168（再培训局，FGS）。艾里逊L.施佩尔是一个大学的外科医生学会爱惜学者。 Yashuhiro虎岛是由一家儿童医院洛杉矶萨班研究所的研究生涯发展奖。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| HBSS | Gibco | 114170-112 | |
| Antibiotic-Antimycotic 100X | Invitrogen | 15240-062 | |
| Dispase | Gibco | 17105-041 | |
| Collagenase Type 1 | Worthington | LS004194 | |
| DMEM High Glucose 1X | Gibco | 11995-065 | |
| Heat inactivated FBS | Invitrogen | 16140-071 | |
| Biofelt 100% PGA | Concordia Medical | FELT01-1005 | For polymer preparation as in Ref. 4 |
| Poly-L-lactic acid | Durect | B6002-1 | For polymer preparation as in Ref. 4 |
| Type I Collagen, rat tail | Sigma-Aldrich | C3867-1VL | For polymer preparation as in Ref. 4 |
| Ketoprofen 100 mg/ml | Fort Dodge Animal Health | 71-KETOI-100-50 | |
| LabDiet 5001 rodent chow | LabDiet | 5001 | |
| Septra 200 mg / 40 mg per 5 ml, USP | Hi-Tech Pharmacal | 50383-824-16 | |
| Isoflurane, USP | Phoenix Pharmaceuticals | 57319-507-06 |
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Tags
IntestineMurine ModelTESISmall Bowel ResectionShort Bowel SyndromeFunctional Malabsorptive StateMorbidityMortalityHealthcare CostsParenteral Nutrition DependenceLiver FailureCirrhosisMultivisceral Organ TransplantationMulticellular Organoid Units-on-scaffold ApproachMucosal ElementsMesenchymal ElementsIntestinal Stem Cell NicheTransgenic ToolsImmunocompromised Mouse StrainsMouse Xenograft FormationGood Manufacturing Practice (GMP) Reagents
Citer Cet Article
Barthel, E. R., Speer, A. L., Levin, D. E., Sala, F. G., Hou, X., Torashima, Y., Wigfall, C. M., Grikscheit, T. C. Tissue Engineering of the Intestine in a Murine Model. J. Vis. Exp. (70), e4279, doi:10.3791/4279 (2012).