上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架
Summary
This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.
Abstract
该协议细节无细胞，但生物功能，肾细胞外基质（ECM）的支架是小规模的模型底物器官规模组织发育有用的产生。只SD大鼠肾脏通过将导管插入肾动脉插管和灌注一系列低浓度去污剂剂（Triton X-100和十二烷基硫酸钠（SDS））超过26小时，以获得完整的，全肾支架与完整perfusable血管，肾小球和肾小管。以下脱细胞，肾支架放在定制设计的灌注生物反应器容器内，并且所述插入导管肾动脉被连接到包括一个灌注回路：蠕动泵;油管;和任选的探针，用于pH值，溶解氧，和压力。杀菌用过乙酸和乙醇，和平衡pH值（7.4）的支架后，将肾支架是一个LARG内通过培养基的灌注准备播种电子容量培养箱保持在37℃和5％的CO 2。通过肾动脉15分钟减少流至生理速率前四千万肾皮质小管上皮（RCTE）细胞注射后，迅速灌注通过下高流动，支架（25毫升/分钟）和压力（〜230毫米汞柱） （4毫升/分钟）。 RCTE细胞主要在肾皮质内填充管状ECM小生，增生，形成小管上皮结构在灌注培养七天。在培养基A 44μM的刃天青溶液通过肾脏中介质交流灌注1小时至小管中提供的细胞生存力和增殖荧光剂，氧化还原类代谢的评估。肾脏灌注生物反应器允许介质的非侵入性采样用于生化评估，和多个入口允许通过肾静脉或输尿管替代逆行播种。这些协议可以用于recellularize肾支架用多种类型的细胞，包括血管内皮细胞，肾小管上皮细胞，和基质成纤维细胞，对本系统内的快速评价。
Introduction
由于患者终末期肾功能衰竭患人数继续增加，存在可供移植的供体肾脏的数量严重和日益短缺。不能以满足不断上升的一些候选人的需求等待上市的肾移植，促使研究肾器官工程与开发定制，植入的肾脏移植需求1,2的终极目标。构建从患者自身的细胞功能肾组织将消除需要终身免疫抑制，减少患者的时间花费在透析等待肾移植的数量，并延长救命移植到更多的患者有慢性肾脏疾病。
朝向生物工程使用源自患者的细胞中的肾组织的第一步是建立一个支架，作为一个支持基板肾实质（ 例如管状上皮的Lial），间质成纤维细胞和血管细胞生长。从天然器官的细胞外基质（ECM的）生物材料衍生支架有几个特点，使它们希望在组织工程，其中包括他们的天然生物成分的使用;适当的宏观和微观结构赋予的生理功能;和细胞生物相容性，促进细胞粘附，迁移和建设性的组织重构3。一个有前途的方法生产的支架为肾组织再生是通过同种异体或异种肾脏脱细胞，同时保留大部分的肾脏ECM 4复杂天然蛋白质组合物，保留器官的固有建筑复杂，并克服与自下而上关联的难度了厚厚的细胞化组织提供血管供应制定细胞支架recellularization 5后工程。
灌注脱细胞是一个过程其中去污剂，酶，或其它细胞破坏解决方案通过器官6的血管网均匀地输送。这个策略已被确立为一种有效的方法，以获得无细胞器官的基于ECM的支架为三维（3D），用于全器官工程6-8生物模板，就证明了无细胞肾模板从废弃人的肾脏9的开发从大的动物（ 如猪10，山羊11）和啮齿动物来源获得12异种肾脏。特别是，使用小动物模型如啮齿类需要更少的细胞和培养基，这对于器官recellularization研究中，细胞数目通常是有限的特别有益的，因为与干细胞衍生的组织的情况下。所描述的脱细胞协议的目标是生产无细胞的肾细胞外基质，可以用来作为3D脚手架系统为肾脏structur的再生上课，包括对重新填充本例与人肾皮质小管上皮（RCTE）细胞在肾小管。我们先前描述的一个最佳的，基于洗涤剂的大鼠肾脱细胞协议7，其较为迅速（约1天）比以前报道的其它方法的我们的严格的评价（Ross 等 .-5天12，Song 等人 .-4.5天13），并暴露了器官的变性剂十二烷基硫酸钠（SDS）的相当低的浓度（0.1％）脱细胞比以前的报告12-15中。
研究数量有限所描述的使用灭鼠肾脏的脱细胞和随后用作3D支架的细胞再增殖的12-16（1别处审查）。在这个协议中，我们提供了先前建立的，最佳的脱细胞的策略的详细说明用于制备无细胞肾支架从只SD大鼠肾脏7。使用自定义设计的双能播种和维护灌注培养17灌注生物反应器，我们recellularize脱细胞支架肾脏与人体RCTE细胞，重新填充一致管状部件在这些脱细胞基质，增殖，而在灌注培养存活了一个多星期。我们进一步证明我们使用刃天青灌注试验的-一种廉价的，无细胞毒性的，非侵入性的代谢评估先前用于毒性研究17 -随时间7提供的recellularized肾脏内细胞的活力和增殖的指示。
Protocol
Representative Results
Discussion
所描述的脱细胞协议始终如一地产生完全无细胞肾细胞外基质充当3D模板人肾皮质小管上皮细胞（近端和远端小管衍生）的培养中，除了血管内皮细胞7,17。管状肾血管用作均匀输送试剂和细胞的整个支架内的生物反应器设置的关键特征，因此能够使灌注脱细胞，细胞接种，长期灌注培养，和刃天青灌注协议。如肾动脉之前器官灌注等，适当的插管是至关重要的，必须采取特别小心，以确?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank the support of the Zell Family Foundation. We recognize support from the Northwestern Memorial Foundation Dixon Translational Research Grants Initiative, the American Society of Transplant Surgeon’s Faculty Development Grant, and a Research Grant for the Young Investigator from the National Kidney Foundation of Illinois. We acknowledge support from the Robert R. McCormick Foundation. This work was also supported by NIDDK K08 DK10175 to J.A.W. Imaging and histology cores used for this research is supported by the Mouse Histology and Phenotyping Laboratory, Electron Probe Instrumentation Center (EPIC), and Simpson Querrey Institute Equipment Core at Northwestern University, and a Cancer Center Support Grant (NCI CA060553). The authors would like to acknowledge the Northwestern University Microsurgery Core for rodent kidney procurements. Evaluation of renal tubular epithelia morphology following recellularization conducted in the Fluorescence Microscopy Shared Resource supported by P30 CA118100.
Materials
| Reagents | |||
| TRITON X-100, Proteomics Grade, AMRESCO | VWR | M143-4L | |
| Sodium dodecyl sulfate solution, BioUltra, for molecular biology, 20% in H2O | Sigma-Aldrich | 05030 | |
| Peracetic acid solution, purum, ~39% in acetic acid (RT) | Sigma-Aldrich | 77240 | Peracetic is flammable and corrosive. Prepare within a fume hood using appropriate personal protective equipment (e.g. gloves, goggles). |
| 200 proof ethanol | VWR | V1001TP | |
| Sigmacote, siliconizing reagent for glass and other surfaces | Sigma-Aldrich | SL2 | For treatment of bioreactor reservoirs. Referred to in text as siliconizing reagent. |
| DMEM/F12 media | Life Technologies | 11320-033 | |
| Corning cellgro Fetal Bovine Serum Premium, Mediatech | Corning | 35-010-CV | |
| Penicillin-Streptomycin Solution, 100X 10,000 I.U. Penicillin 10,000 µg/mL Streptomycin | Corning | 30-002-CI | |
| TrypLE Express (1X), Phenol Red | Life Technologies | 12605-028 | Referred to in text as cell dissociating enzyme. |
| Trypan Blue Stain (0.4%) | Life Technologies | 15250-061 | |
| Resazurin sodium salt | Sigma-Aldrich | R7017 | |
| Equipment: | |||
| Masterflex L/S Digital Drive, 600 RPM, 115/230 VAC | Cole-Parmer | EW-07522-20 | |
| Masterflex L/S large cartridges for 07519-05 and -06 pump heads. | Cole-Parmer | EW-07519-70 | Referred to in text as large pump cartridge. |
| Masterflex L/S 8-channel, 4-roller cartridge pump head. | Cole-Parmer | EW-07519-06 | |
| Straight 6" specimen forceps, serrated | VWR | 82027-438 | |
| *Kidney perfusion bioreactor | WilMad Labglass | *Custom designs | Bioreactors are produced as described by WilMad Labglass. The designs have been described in depth in a previous publication. |
| Perfusion Circuit Components | |||
| 24 G x 0.75 in. BD Insyte Autoguard shielded IV catheter (0.7 mm x 19 mm) made of BD Vialon biomaterial. Has notched needle. (50/sp, 200/ca) | BD Biosciences | 381412 | Referred to in text as 24 gauge catheter. |
| Masterflex PharMed BPT Tubing, L/S #14, 25' | Cole-Parmer | HV-06508-14 | Referred to in text as peristaltic pump tubing. |
| Peroxide-Cured Silicone Tubing, 1/16"id X 1/8"OD, 25 Ft/pack | Cole-Parmer | HV-06411-62 | Referred to in text as 1/16" ID silicone rubber tubing. |
| Masterflex platinum-cured silicone tubing, L/S 14, 25 ft. | Cole-Parmer | HV-96410-14 | Referred to in text as thick-walled 1/16" ID silicone rubber tubing. |
| VWR Silicone Tubing, 1/4" ID x 0.5" OD | VWR | 89068-484 | |
| Acro 50 Vent Filters, Pall Life Sciences | VWR | 28143-558 | Referred to in text as 0.2 micron vent filter. |
| Cole-Parmer Luer Adapters, Male Luer Lock to 1/8" ID, Nylon, 25/Pk | Cole-Parmer | T-45505-11 | Referred to in text as male Luer lock to 1/8" barbed adapter. |
| Cole-Parmer Luer Accessory, Male Luer Plug, Nylon, 25/Pk | Cole-Parmer | EW-45505-58 | |
| Female luer x female luer adapter, Nylon, 25/pk | Cole-Parmer | EW-45502-22 | Referred to in text as female Luer x female Luer adapter. |
| Cole-Parmer Luer Accessory, Female Luer Cap, Nylon, 25/Pk | Cole-Parmer | EW-45502-28 | |
| Smiths Medical Large Bore Hi-Flo Stopcocks # MX4311L – 3-Way Hi-Flo Stopcock with Extended Male Luer Lock, Non-DEHP Formulation, Latex Free (LF), Lipid Resistant, Non-PVD, 50/cs | Careforde Healthcare | 10254821 | Smiths Medical (vendor) catalogue number is #MX4311L. |
| Other Components | |||
| 5 mL BD Luer-Lok disposable syringe with BD Luer-Lok™ tip. | BD Biosciences | 309646 | |
| 35 mm x 10 mm Easy Grip Culture Dish | BD Biosciences | 353001 | Used to draw cell suspension into syringe for cell seeding. |
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