小鼠和人源性胃上皮单层培养对再生的研究
Summary
在这里, 我们描述了建立和培养人和老鼠衍生的3维 (3D) 胃 organoids 的协议, 以及将 3D organoids 转移到2维单层的方法。本文还介绍了用胃上皮单层作为再生研究的一种新的擦伤创面检测方法。
Abstract
体外对胃创面修复的研究通常涉及使用胃癌细胞系进行细胞增殖和迁移的刮伤试验。然而, 这种化验的一个关键限制是它们的细胞类型的同源分类。修复是一个复杂的过程, 需要几个细胞类型的相互作用。因此, 研究一种缺乏细胞亚型的文化, 是我们理解修复过程必须克服的一个问题。胃 organoid 模型可以缓解这个问题, 即细胞类型的异构收集密切反映的胃上皮或其他本机组织在体内。这里演示的是一本新颖的,体外刮伤试验, 从人或小鼠3维 organoids, 然后可以转移到胃上皮单层作为完整的 organoids 或作为一个单独的细胞悬浮分离organoids。该协议的目的是建立 organoid 的胃上皮膜, 可用于一个新的擦伤伤口检测系统, 以研究胃再生。
Introduction
使用刮伤检测是研究修复和再生的常用方法1,2,3,4,5,6。建议的方法可用于专门研究胃再生和幽门螺杆菌的殖民化。过去, 胃癌细胞系被用来研究幽门螺杆菌 (幽门螺杆菌) 感染 1,2和直到最近的胃癌细胞株, 如 AGS 细胞被青睐1 ,2,3,4。胃癌细胞培养的一个局限性是它们未能重述胃上皮细胞的多样性。为了解决这一局限性, 这里展示了一个主要的人和小鼠3维胃底胃 organoids (FGOs) 的建立和转移的胃上皮单层的伤口愈合检测的基础上改进方法由 Schlaermann et al描述。7我们演示了从剪切后的3D 胃 FGOs 或 FGO 衍生的单细胞中提取的胃上皮细胞, 保留了一种极化的蜂窝状成分, 它与胃上皮的在体内有密切的关系。鉴于胃癌细胞系并没有证明这种技术所显示的细胞组成, 目前的协议比其他的擦伤伤口检测方法有优势。这种方法已被优化, 使单分子膜可以建立从整个 organoids 或从一个单一的细胞悬浮从分离 organoids 和电镀使用基底细胞膜基质或胶原涂层。该协议的总目标是建立一个 organoid 的胃上皮单层培养的新的伤口愈合试验。
Protocol
为了避免污染, 在无菌组织培养罩内全部执行协议。根据经批准的辛辛那提大学 irb 协议 (irb 协议号: 2015-4869), 从患者的袖状胃切除术中收集人体胃底组织。所有老鼠的研究都得到了辛辛那提大学机构动物保育和使用委员会 (IACUC) 的批准, 它维持着美国实验室动物护理 (AAALAC) 设施的评估和认可协会。 1. 建立人源性3D 胃胃底 Organoids 注意: 此协议基于以前在?…
Representative Results
Organoids 是从人的主体/身体或鼠胃组织的眼底 (图 1A) 派生的。在胶原酶或 EDTA 消化的人或老鼠的胃组织分别后, 腺体埋入基底膜基质和培养 6-7 天 (图 1A)。图 1B演示了人源性胃 organoids (huFGOs) 的形成, 然后将其转移到基底膜基质涂层室幻灯片上的胃上皮层 (huGEM) 上。经过4天的文化, 汇合 huGEMs 被用于刮伤化验…
Discussion
本议定书详细说明了建立人和老鼠的胃上皮膜, 可用于刮伤检测。该协议依赖于从原始人 (或小鼠) 组织中分离出的干细胞的概念, 是由 Schlaermann et al7首次发布的修改后的协议。特别是, 我们已经优化的协议, 以建立一个汇合和极化胃上皮单层, 表达的主要细胞血统, 可以发现在胃上皮内在体内。胃溃疡修复是一个复杂的过程, 涉及组织重新上皮, 再生和扩散<sup class="xref…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到 NIH (NIDDK) 5 R01 DK083402-07 补助金 (YZ) 的支持。
Materials
| Advanced Dulbecco's modified Eagle/F12 medium (basal media) | Life Technologies | 12634-010 | |
| GlutaMAX (L-glutamine) | Life Technologies | 25030-081 | |
| Penicillin/Streptomycin | Thermo Scientific | SV30010 | |
| Amphotericin B/Gentamicin | Thermo Scientific | R01510 | |
| Kanamycin | Thermo Fisher | RO1510 | |
| HEPES Buffer | Sigma Aldrich | H0887 | |
| n-Acetylcystine | Sigma Aldrich | A9165 | |
| N2 | Life Technologies | 17502-048 | |
| B27 | Life Technologies | 12587-010 | |
| BMP Inhibitor (Noggin) | Pepro Tech | 250-38 | |
| Gastrin | Tocris | 3006 | |
| EGF | Pepro Tech | 315-09 | |
| FGF10 | Pepro Tech | 100-26 | |
| Nicotinamide | Sigma Aldrich | N0636 | |
| Y-27632 ROCK Inhibitor | Sigma Aldrich | Y0503 | |
| TGF-β Inhibitor | Tocris | 2939 | |
| Ca2+/Mg2+ -free DPBS | Fisher | 21-031CV | |
| Dulbecco's modified Eagle Medium (DMEM) | Life Technologies | 12634-010 | |
| Fetal Bovine Serum | FBS | ||
| OPTIMEM | Invitrogen | ||
| 0.22µM filter | Fisher | 099-720-004 | |
| 37 °C Water Bath | |||
| Collagenase Type 1 | Worthington | LS004214 | |
| Bovine Serum Albumin | Sigma Aldrich | A9418 | |
| Kimwipes (tissue paper) | Fischer Scientific | 06-666C | |
| 125 mL round bottom flask | |||
| 1 in (25 mm) stir bar | |||
| Rubber Septa | |||
| Sterile Gauze | |||
| Stir Plate | |||
| Ring Stand | |||
| Ring Stand Clamps | |||
| Sterile petri dish | |||
| 18 G needles of 1.5 in length | Thermo Fisher | 305196 | |
| 20 G spinal needles of 3.5 in length | Thermo Fisher | 405182 | |
| 12-well cell culture treated plate | Midwest Scientific | 92012 | |
| Growth Factor Reduced, Phenol Red-free MatrigelTM (Basement membrane matrix) | Fisher | CB-40230C | |
| Sterile Razor Blades | |||
| Wide-tip tweezers | |||
| Curved Hemostatic Forceps | |||
| 200 µL wide pipette tips | Thermo Fisher | 02-707-134 | |
| 5 mL cell culture test tubes | Fisher | 14-956-3C | |
| Oxygen Tank with rubber hosing | |||
| 1 g D-Sorbitol | |||
| Sucrose | Fisher | SS-500 | |
| Dissecting microscope | |||
| Dissecting Tray | |||
| Rocking Table | |||
| Rat Tail Collagen Type 1 | Life Technologies | A10483-01 | |
| Cell culture grade glacial acetic acid | Fisher | A38-212 | |
| Cell culture grade water | Corning | 25-055-CV | |
| 2-well chamber slide | Thermo Fisher | 155380 | |
| 12-well Transwell (polyester membrane inserts) | Corning | 3460 | |
| Cell scraper | Corning | 353086 | |
| Accutase (cell detachment solution) | Stemcell Technologies | 7920 | |
| 263/8 G syringe | Thermo Fisher | 309625 | |
| Razor blade | |||
| L Cells | L cells, a Wnt3a producing cell line, were received as a gift from Dr. Hans Clevers (Hubrecht Institute for Developmental Biology and Stem Cell Research, Netherlands). | N/A | |
| HEK-293T Rspondin secreting cells | A modified HEK-293T R-spondin secreting cell line was obtained from Dr. Jeff Whitsett (Section of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children's Hospital Medical Centre and The University of Cincinnati College of Medicine, Cincinnati, USA ). | N/A | |
| HK-ATPase Primary Antibody | Invitrogen | SC-374094 | |
| E-Cadherinn | SantaCruz | sc-59778 | |
| UEA1 | Sigma Aldrich | L9006 | |
| Hoechst 33342 | Thermo Fisher | H3570 | |
| Alexafluor 488 secondary antibody (Donkey anti-mouse 488 secondary antibody) | ThermoFisher Scientific | R37114 |
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Citazione di questo articolo
Teal, E., Steele, N. G., Chakrabarti, J., Holokai, L., Zavros, Y. Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration. J. Vis. Exp. (135), e57435, doi:10.3791/57435 (2018).