原发性肾组织段的显微解剖与新型无支架结构技术的结合
Summary
组织工程的肾脏结构提供了解决的器官短缺和有害影响的透析。在这里, 我们描述了一个小鼠肾脏的显微解剖的协议, 以隔离皮质髓质段。这些片段植入无支架细胞结构, 形成肾 organoids。
Abstract
肾脏移植现在是治疗终末期肾脏疾病的主流疗法。然而, 约有9.6万人在等待名单上, 只有1/4 的患者实现移植, 迫切需要为那些器官衰竭者提供替代品。为了减少透析带来的有害后果, 以及它所招致的整体医疗费用, 正在积极调查寻找移植器官的替代办法。植入性组织工程的肾细胞结构是一种替代失去的肾脏功能的可行方法。这里, 第一次被描述, 是小鼠肾脏的显微解剖为分离生活皮质髓质肾脏片断。这些片段能够迅速纳入无支架的内皮纤维细胞结构, 这可能使与宿主血管的快速连接一旦植入。成年小鼠肾脏是从活体捐献者那里获得的, 其次是立体镜显微解剖, 得到 200-300 µm 直径的肾段。多肾结构是用从一肾中收获的原发性肾段制作而成。该方法证明了一种可以从器官中抢救功能性肾组织的程序, 否则将被丢弃。
Introduction
慢性肾脏疾病 (CKD) 是当前全球主要的公共卫生挑战之一1。在美国, CKD 患病率超过总人口的 14%, 超过60万的美国人患有最严重的疾病, 终末期肾病 (ESRD)2。目前的治疗方案可供那些 ESRD 包括透析和肾移植。虽然每年约有2.5万名患者接受肾移植, 但每年有大量患者被增加, 导致等待救生器官和接受移植的人之间的巨大差距3。除了对长寿和生活质量造成严重的负面影响外, 透析还带来了惊人的经济负担。在 2014年, 医疗保险支付的索赔总额超过300亿美元的 ESRD 患者2。由于器官供应有限, 需要透析的患者没有明显的下降趋势, 因此, 旨在确定透析和移植的替代解决方案的研究工作一直是重要的。即使是相对较短的延迟, 透析的需要增加了病人的质量调整生活年限和生产力的大量, 而推迟透析相关费用4,5,6。
在组织工程和再生医学实验室中, ESRD 的功能性组织丧失的解决方案目前正在研究中, 从脚手架为基础的 organoid 制造到整个器官工程都有广泛多样的方法。瓣膜细胞植入的器官结构7,8,9,10,11。综述复杂的肾脏结构从边缘或被放弃的肾脏仅部份地被调查了。事实上, 为移植而采购的肾脏的近20% 因各种原因被丢弃,12,13。这些假定的移植物的功能性肾脏组织可以被利用并纳入一个或多个组织工程结构。先前的研究表明, 与这些被丢弃的器官合作的可行性, 利用肾脏为组织工程目的的额外细胞矩阵14,15。然而, 很少有使用健康肾脏的主要 nephronal 组织为组织工程目的16,17,18。
Kim et以前描述的一种方法是将肾脏 “段” 从健康的大鼠肾脏中分离出来, 然后在羟基酸 (PGA) 支架上播种, 用于构造制造16。然而, 关于精确解剖方法的信息很少, 从精细切碎和过滤的结合中得到了部分。我们描述了这个协议的修改, 这同样产生了离散的肾段与完整的 nephronal 体系结构, 而是依靠显微切割技术。切除是对活体成年小鼠进行的, 然后将肾脏转移到切除肾囊的解剖显微镜, 并进一步解剖组织。小孔 30½ G 针作为切割仪器, 也作为辅助解剖辅助, 因为针直径等于肾段的靶直径。这种孤立的, 在这种情况下, 小鼠, 肾部分保持在培养的生存能力, 并纳入无支架内皮细胞成纤维细胞结构19。这些构造以前被用来设计其他器官, 包括生物人工胰腺20。
Protocol
所有的动物外科手术程序都是在南卡罗来纳州医科大学的机构动物护理和使用委员会 (IACUC) 批准的, 然后再进行任何动物手术或任何动物组织的使用。 1. 小鼠肾切除术 不要用手术面罩和蓬松帽来减少污染的风险。在手术区的设置中保持不孕。 将非 fenestrated 的手术窗帘放在手术台上。 在无菌窗帘上打开一包蒸压仪器。肾切除术所需的器械包括3小止血剂?…
Representative Results
所描述的协议产生大约50个肾脏片段每锥体2毫米3段肾脏组织。已处理和成像的肾段有不同比例的管状和肾小球成分 (请参见图 2)。完整的片段进行了化验, 以确定每24小时的不同片段的生存时间三天。绿色荧光 calcein 是存在的细胞内酯酶活性, 指示的活细胞。红荧光溴 homodimer-1 被认为是由于等离子膜完整性的丧失。虽然这些片段是完整的, 三?…
Discussion
用于设计活体肾脏组织结构的方法在所使用的细胞类型和生物材料方面差别很大, 在许多情况下, 在文献7中已经过时或没有很好的特征。虽然许多人使用干细胞方法或综述独立的肾脏结构的个别成分, 但从细胞悬浮中人工再造26多个不同分化细胞类型的整个器官的前景是压倒性地考虑21。这很可能是其他人在组织工程应用中使用肾组织片段的方法。如上所述, 以前…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
NIH 机构博士后培训补助金, NIH-HL-007260
Materials
| Non-fenestrated Sterile Field | Busse Hospital Disposables | 696 | |
| Fenestrated Sterile Field | Busse Hospital Disposables | 697 | |
| Halsted Mosquito Forceps 5 Curved | Miltex | Mil-7-4 | "Hemostat" in manuscript |
| Extra Fine Graefe Forceps, Curved with teeth | Fine Science Tools | 11155-10 | Fine forceps with teeth |
| Extra Fine Graefe Forceps, Serrated (without teeth) | Fine Science Tools | 11152-10 | Fine forceps without teeth |
| Fine Scissors – Tungsten Carbide | Fine Science Tools | 14568-09 | Iris Scissors |
| Betadine Surgical Scrub with Pump, Povidone-iodine 7.5% | Purdue Products L.P. | 67618-151-17 | |
| Sterile Cotton Gauze Pad (4" x 4") | Fisher Healthcare | 22-415-469 | |
| Dulbecco's Phosphate Buffered Solution | Corning | 21-030-CV | |
| Penicillin/Streptomycin Solution, 100X | Corning | 30-002-Cl | |
| Isoflurane, USP | Manufacturer: Piramal, Distributor: McKesson | 2254845 | |
| Nair Hair Remover | Nair | 22600-23307 | Hair Removal Cream in text |
| 200 Proof Ethanol | Decon Laboratories | 2705 | Diluted to 70% Ethanol Solution |
| BioLite 60mm Tissue Culture Dish | Themo-Scientific | 130181 | |
| Press'n Seal | Glad | 12587-70441 | Applied to Stereoscope |
| SZX16 Stereo Microscope | Olympus | SZX16 | |
| Fiber Optic Illuminator | Cole Parmer | 41720-20 | |
| Self-Supporting Dual-Light Pipe, 23" L Gooseneck | Cole Parmer | EW-41720-60 | |
| Scalpel Handle #3 | Miltex | Mil-4-7 | |
| Sterile Rib-Back Carbon Steel Blade, Blade Size 15 | Bard-Parker | 371115 | |
| 31 1/2 Gauge Needle | ThermoFisher Scientific | 14-826F | Becton Dickinson 305106 |
| Dulbecco's Modified Eagle's Medium | Corning | 10-017-CV | |
| Fetal Select 100% Bovine Serum | Atlas Biologicals | FS-0500-AD | |
| Normal Human Dermal Fibroblasts | Lonza | CC-2511 | |
| Human Adipose Microvascular Endothelial Cells | Sciencell Research Laboratories | 7200 | |
| Surgical Loupes (2.5x) | Orascoptic | (N/A) Custom Order | |
| FGM-2 (Fibroblast Basal Medium with FGM-2 SingleQuots Added) | Lonza | CC-3131, CC-4126 | |
| EGM-2 (Endothelial Basal Medium with EGM-2 SingleQuots Added) | Lonza | CC-3156, CC-4176 | |
| Live/Dead Viability/Cytotoxicity Kit for Mammalian Cells | ThermoFisher Scientific | L3224 | |
| Anti-Cytokeratin-18 Antibody | Abcam | ab668 | |
| Goat anti-Mouse IgG, Alexa Fluor 633 | ThermoFisher Scientific | A-21052 | |
| Goat anti-Rabbit IgG, Alexa Fluor 546 | ThermoFisher Scientific | A-11010 | |
| Anti-Von Willebrand Factor Antibody | Abcam | ab6994 | |
| Albumin, Fluorescein isothiocyanate Conjugate | Sigma Aldrich | A9771-50MG | |
| Hoescht 33342 | BD Pharmingen | 561908 | |
| Background Buster | Innovex Biosciences | NB306 |
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Diesen Artikel zitieren
Arbra, C. A., Nadig, S. N., Dennis, S. G., Pattanaik, S., Bainbridge, H. A., Rhett, J. M., Fann, S. A., Atkinson, C., Yost, M. J. Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology. J. Vis. Exp. (133), e57358, doi:10.3791/57358 (2018).