组织工程应用中植物组织去细胞的两种方法
Summary
在这里, 我们介绍, 并对比两个协议用于 decellularize 植物组织: 洗涤剂为基础的方法和无洗涤剂的方法。这两种方法都留下了植物组织的细胞外基质, 然后可以用作组织工程应用的支架。
Abstract
目前用作组织置换支架的自体、人工合成和动物衍生的移植物由于可用性低、生物兼容性差和成本高而受到限制。植物组织具有良好的特性, 使其独特的适合作为脚手架, 如高表面积, 优良的水传输和保留, 相互连通的孔隙率, 现有的血管网络, 和广泛的机械性能。本文介绍了两种用于组织工程应用的植物去细胞的成功方法。第一种方法是基于洗涤剂浴去除细胞物质, 这与以前建立的用于清除哺乳动物组织的方法相似。第二种是一种无洗涤剂的方法, 它适应于一种隔离叶脉的协议, 包括使用加热漂白剂和盐浴清除叶子和茎。这两种方法都能产生类似机械性能和低细胞代谢影响的脚手架, 从而使用户能够选择更适合其预期应用的协议。
Introduction
组织工程出现在二十世纪八十年代, 以创建活体组织替代品, 并有可能解决重大器官和组织短缺1。一种策略是使用支架刺激和引导身体再生缺失的组织或器官。尽管先进的制造方法, 如3维印刷生产的脚手架具有独特的物理性能, 但能够制造具有多种可实现的物理和生物特性的脚手架仍然是一个挑战2,3. 此外, 由于缺乏功能性血管网络, 这些技术在再生3维组织方面受到限制。使用瓣膜动物和人体组织作为支架帮助规避此问题4,5,6,7。但是, 高成本、批处理可变性和有限的可用性可能限制了瓣膜动物支架的广泛使用8。还有人担心潜在的疾病传播给病人和免疫反应的一些瓣膜哺乳动物组织9。
纤维素, 来源于植物和细菌来源, 已广泛用于产生生物材料的广泛应用于再生医学。一些示例包括: 骨骼10、11、软骨12、13、14和伤口愈合15。由纤维素组成的脚手架有一个额外的好处, 因为它们是耐用的, 并耐被哺乳动物细胞分解。这是由于哺乳动物细胞不产生分解纤维素分子所必需的酶。相比之下, 使用大分子从胞外基质 (如胶原蛋白) 生产的脚手架容易被分解为16 , 可能不适合于长期应用。胶原支架可以通过化学交联来稳定。然而, 由于影响到支架的生物相容性的交叉连接器的固有毒性, 有一个权衡 17.反之, 纤维素有可能长期留在植入地点, 因为它不受哺乳动物细胞的酶降解18,19,20。通过水解预处理和与多种纤维素酶21 的协同提供, 可以改变降解速率。在小鼠22的研究中也证明了瓣膜植物衍生纤维素支架的生物相容性在体内。
经过数以百万年的进化, 植物改良了它们的结构和组成, 以提高流体输送和保持的效率。植物血管容器通过分枝成较小的血管来最小化水力阻力, 类似于根据默里定律23的哺乳动物血管。去细胞后, 该植物的复杂网络的船只和互联毛孔保持。考虑到大量不同种类的植物, 植物衍生的支架有可能克服目前影响组织工程中支架的设计限制24,25。例如, Modulevsky et表明, 当瓣膜苹果组织植入在鼠标22的背面时, 血管生成和细胞迁移发生。同样, Gershlak et表明, 内皮细胞可以在瓣膜叶的血管内生长24。在一个单独的实验中, Gershlak et 等人也能够表明心肌细胞可以生长在叶子表面, 并且能够收缩24。
植物还包括从细胞到宏观尺度的复杂组织, 即使用迄今为止开发的最先进的制造技术, 也很难实现。植物组织的复杂层次设计使它们比其成分的总和26强。植物拥有大量不同的机械性能, 从刚性和坚韧的成分, 如茎, 更灵活和柔韧的, 如叶27。叶片的大小、形状、断裂强度、血管化程度等不同种类的不同, 并可进行多种亲水性。总的来说, 这些植物的特性表明, 瓣膜植物可以作为独特和功能强大的医疗设备, 包括组织工程支架。
本议定书的重点是两种方法, decellularize 植物组织, 如叶和茎, 用作支架在组织工程。第一种方法是以洗涤剂为基础的技术, 使用一系列的浴池来去除 DNA 和细胞物质, 这已经从广泛使用的技术 decellularize 哺乳动物和植物组织中进行了调整6,22,25 ,28,29,30。第二种方法是无洗涤剂的, 它适应于通常用于去除叶子的软组织的 “骨架” 协议31。先前的工作表明, 在漂白和碳酸氢钠溶液中煨的叶子促进了从周围的软组织分离的血管31。该技术可引回到 17th和 18th世纪进行的实验, 例如 Albertus 塞巴32和爱德华帕里什33的工作。这些实验围绕着离开植物物质, 如叶子和水果, 在水中浸泡了很长一段时间 (几周到几个月), 让柔软的组织自然腐烂。这里的 “骨架” 方法适合使用较温和的条件, 如较长的潜伏期在较低的温度, 以消除细胞残留, 并避免严重扰乱软组织结构。在此详细的实验中, 使用了三种植物类型:榕刚毛、 Pachira aquatica和一个藤黄的种类。对这两种方法的 DNA 定量、力学测试和对细胞代谢活动的影响进行了描述。
Protocol
Representative Results
Discussion
本文介绍了两种 decellularize 植物组织的方法。这里提出的结果, 加上先前研究的结果25, 表明所提出的协议可能适用于广泛的植物种类, 可以在茎和叶上进行。这些程序是简单的, 不需要专门的设备, 所以植物去细胞可以在大多数实验室进行。值得注意的是, 去细胞后, 支架必须具有功能性, 以促进哺乳动物细胞黏附。不同的功能化技术, 使哺乳动物细胞黏附和生长的植物组织已被描?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们要感谢奥尔布里希花园的约翰 Wirth 慷慨地提供了这个项目使用的标本。这项工作由国家心脏、肺和血液学会部分支持 (R01HL115282 到 G.R.G.)国家科学基金会 (DGE1144804 和 G.R.G.), 威斯康星大学外科和校友基金 (H.D.L.)。这项工作也部分由环境保护局 (83573701 号星赠款)、国立卫生研究院 (R01HL093282-01A1 和 UH3TR000506) 和国家科学基金会 (IGERT DGE1144804) 支持。
Materials
| Sodium dodecyl sulfate | Sigma Life Science | 75746-1KG | |
| Triton X-100 | MP Biomedicals, LLC | 807426 | Non-ionic surfactant referenced in paper. Very viscous reagent, can help to cut end of pipette tip when drawing it up. |
| Concentrated bleach (8.25% sodium hypochlorite) | Clorox | Item #: 31009 | Standard concentrated bleach. |
| Sodium bicarbonate | Acros Organics | 217120010 | Can be substituted with sodium hydroxide or sodium carbonate. |
| 8 mm Biopunch | HealthLink | 15111-80 | Cuts samples that fit well in 24 well plate |
| Belly Dancer-Shake table | Stovall Life Sciences | BDRAA115S | Use low speeds to not damage tissues. Can use any model/brand of shake table. |
| Isotemp hot/stir plate | Fisher Scientific | Can use any style/brand of hot/stir plate. | |
| Beaker | Any | Can use any size beaker as long as it will fit your samples and not overcrowd them. | |
| Tris Hydrochloride | Fisher Scientific | BP153-500 | |
| DMEM | Corning | MT50003PC | |
| Quant-iT Picogreen dsDNA assay | Life Technologies | P11496 | Can use any dsDNA quantification mehtod on hand. |
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