该<em>离体</em>文化与模式识别小鼠肠道组织体受体刺激
Summary
这里，收获，维护和治疗与病原体相关的分子模式（PAMP），并李斯特菌小鼠小肠组织体的协议被描述，以及强调基因表达和蛋白质正确规范化技术。
Abstract
主肠组织体是具有显著影响粘膜免疫学领域的潜力的一个有价值的模型系统。然而，的类器官生长特性的复杂性进行了调查显著警告。具体而言，每个单独的器官样的生长模式是高度可变的，并创建在培养的上皮细胞的异质群体。有了这样的警告，共同组织培养做法不能简单应用到类器官系统，由于蜂窝结构的复杂性。计数和电镀对细胞数，这是单独分离的细胞，如细胞系共同仅基于，不是组织体的可靠方法，除非被施加一些正常化技术。正火总蛋白含量因驻地蛋白基质制成复合物。在细胞数量，形状和细胞类型而言，这些特征应在评估分泌CON时加以考虑从类器官质量帐篷。该协议已经产生勾勒出一个简单的程序，以文化和治疗小肠类器官与病原微生物和病原相关分子模式（PAMP）。它还强调当蛋白质分析被这样的挑战之后进行应该应用的标准化技术。
Introduction
的能力，以收集和小肠，结肠，胰腺，肝脏和脑培养初级组织体已经描述并有密切关系理解一个多种生理学代表现象为组织生物学1-5令人兴奋的进展。描述小肠类器官培养和维护的方法，首次报道了佐藤等人出来汉斯Clevers 1的实验室。在此之前的方法，收获和证明原肠上皮细胞的培养在限于并在维持上皮细胞的生长是无效的。方法包括通过用酶，例如胶原酶和分散酶，这将最终导致混合初级成纤维细胞6的生长培养组织的离解。这些条件也将时间在维持上皮细胞培养物的限制。最小的无上皮细胞小生境将形成，因为上皮细胞会进入细胞凋亡，由于缺乏适当的生长因子或接触丧失完整性，称为anokis 7。所述3D-类器官培养系统的出现提供了含有肠细胞类型的光谱中持续培养1培养原肠细胞的方法。这些上皮组织体具有以上的细胞系是它们是由多种分化的细胞的优点，并更好地模仿它们从体内 8衍生的器官。该过程最终“生长在培养皿迷你肠”已被证明是用于评估肠上皮细胞在不同刺激的反应的有价值的工具。调查主肠细胞微生物病原体相关的分子模式（PAMP）的相互作用是相关的免疫学领域，因为这些分子模式可以调节从主机和微生物9多样的响应。不仅可以调查，现在寻求与鼠标类器官这些互动，但他们可以从人类以及2中培养。该技术具有显着改变的个性化医学的潜力，很容易让人猜测的进步，这种技术将在不久的将来成为可能。
该方法的总的目标是提供一种用于培养，扩展的协议，和治疗肠组织体与各种刺激。这种刺激可以最终范围从疫苗，细菌的PAMP，活的病原体，胃肠（GI）和癌症治疗。小鼠肠道组织体的分离和培养是改编自佐藤等人虽然有从原来的方法略有偏差，最终产品是化培养按照本协议时仍然实现。此方法集中在与非同质细胞结构，其必须基于小区n导电的测定时，可以考虑到工作时，描述为合适正常化的适当技术红棕色。
Protocol
Representative Results
Discussion
肠组织体的培养和维护是可以通过用足够的组织培养技术的任何个人掌握的过程。有在传代相比，在更传统的单层生长的细胞时微妙之处，但这些微妙之处不难克服。该方法的关键步骤涉及能够对组织体生长到足够高的密度以获得最佳播种。作为通常可以用细胞系取得了较大的接种密度是不实际的实验，必须缩小与类器官。当有多个处理组，这就变得尤为明显。
该协议的目?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Dr. Sheryl Coutermarsh-Ott, Dylan McDaniel and Bettina Heid for technical discussions. The authors thank Dr. Nanda Nanthakumar for providing the Caco-2 cells. The authors also thank The Multicultural Academic Opportunities Program (MAOP) at Virginia Tech. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Award K01DK092355 (to I.C.A.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Materials
| Fetal Bovine Serum (FBS) | Atlanta Biologicals | S11050 | (Section 1,3,6) Or equivalent brand |
| Sorvall Legend XTR Centrifuge | Thermo | (Section 1,3) | |
| DMEM | GE Healthcare | Sh30243.01 | (Section 1,6) For Caco-2 and HEK293 Rspondin1 cells |
| HEK293T-Rspondin1 secreting cell line | (Section 1) Described and modified from Kim, K.A. et al. Lentiviral particles contained RSPO1(NM_138683) ORF cDNA cloned into a pReceiver-Lv105 backbone custom ordered and purchased from GeneCopoeia. | ||
| 50 ml conical tube | Falcon | 352070 | (Section 1) Or equivalent brand |
| T-175 Flask | Corning | 431079 | (Section 1) Or equivalent brand |
| Protein Matrix | Corning | 356231 | (Section 2,3,4,5,6) Matrigel Growth Factor Reduced |
| HyClone Dulbecco's (DPBS) | GE Healthcare | SH30264.01 | (Section 2,3) |
| DMEM/F12 | Life Technologies | 12634-010 | (Section 2,3) Advanced DMEM/F12 |
| Corning 24 Well TC Plates | Corning | 3524 | (Section 2) |
| N2 Supplement 100x | Life Technologies | 17502-048 | (Section 2) |
| B27 without vitamin A 50x | Life Technologies | 12587-010 | (Section 2) |
| Trizol | Life Technologies | 15596-026 | (Section 2) |
| Glutamine Supplement (Glutamax) | Life Technologies | 35050-061 | (Section 2) Can Combine with Advanced DMEM/ F12 |
| HEPES (1 M) | Life Technologies | 15630-080 | (Section 2) Can Combine with Advanced DMEM/ F12 |
| 10ml Serological Pipet | Falcon | 357551 | (Section 2) Or equivalent brand |
| Murine Noggin | Peprotech | 250-38 | (Section 2) Stock = 100 mg/ml |
| N-Acetyl-L-cysteine | Sigma-Aldrich | A9165 | (Section 2) Stock = 1M |
| Recombinant Mouse EGF | Biolegend | 585608 | (Section 2) Stock = 500 mg/ml |
| Rocker Variable | Bioexpres | (Section 3) | |
| dissecting scissors | (Section 3) | ||
| forceps | (Section 3) | ||
| glass slides | (Section 3) | ||
| dissecting tweezers | (Section 3) | ||
| 25 ml Serological Pipet | Falcon | (Section 3) | |
| EDTA | Sigma-Aldrich | SLBB9821 | (Section 3) 0.5M or alternative TC grade EDTA |
| Sterile Petri Dish 100mm x 15mm | Fisher | FB0875712 | (Section 3) Or equal sized TC dish |
| 1ml Syringe | Becton Dickinson | 309659 | (Section 4) |
| Precision Glide Needle | Becton Dickinson | 305120 | (Section 4) 23G x 1 1/4 (0.6mm x 30mm) |
| Flagellin from Bacillus subtilis | Invivogen | tlrl-bsfla | (Section 5,6) |
| Listeria monocytogenes | ATCC | 19115 | (Section 5,6) (Murray et al.) |
| Hemocytometer | Sigma-Aldrich | Z359629-1EA | (Section 5,6)Or equivalent brand |
| BBL Brain Heart Infusion Agar | Becton Dickinson | 211065 | (Section 5) |
| Bacto Brain Heart Infusion | Becton Dickinson | 237500 | (Section 5) |
| Caco-2 | ATCC | HTB-37 | (Section 6) |
| Trypsin | gibco | 25200056 | (section 6) |
| Methanol | Fisher | A412-4 | (Section 6) |
| SpectraMax M5 | Molecuar Devices | (Section 6) | |
| 96 Well Assay Plate | Corning | 3603 | (Section 6) Black Plate, Clear Bottom TC treated |
| Nuclear Staining Dye | Life Technologies | H1399 | (section 6) Hoechst 33342 |
| T-75 Flask | Corning | 430641 | (Section 6) Or equivalent brand |
| 15 ml conical tube | Falcon | 352096 | (Section1,3) Or equivalent brand |
| 1.7 ml polypropylene tube | Bioexpress | C-3262-1 | Or equivalent brand |
| Quick-RNA MiniPrep | Zymo Research | R1054 | Or equivalent brand |
| TNF-alpha | Applied Biosystems | Mm 00443260_g1 | Taqman gene expression assay kit |
| IL-6 | Applied Biosystems | (Mm 00446190_m1 | Taqman gene expression assay kit |
| IL-1beta | Applied Biosystems | Mm 00434228_m1 | Taqman gene expression assay kit |
| IL-18 | Applied Biosystems | Mm 00434225_m1 | Taqman gene expression assay kit |
| 18s | Applied Biosystems | Hs 99999901_s1 | Taqman gene expression assay kit |
| 7500 Fast Real Time PCR System | Applied Biosystems | ||
| Nexus gradient Mastercycler | Eppendorf | ||
| TaqMan Fast Universal PCR Master Mix | Life Technologies | 4352042 | |
| High Capacity cDNA Reverse Transcription Kit | Life Technologies/Applied Biosystems | 4368814 | |
| Fast Optical 96-Well Reaction Plate, 0.1 mL | Life Technologies/Applied Biosystems | 4346907 | |
| Recombinant Mouse R-Spondin 1 Protein | R&D Systems | 3474-RS-050 | 500 ng/ml |
| chloroform | Sigma-Aldrich | C7559 |
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