由激光蚀刻大师定制的组织培养模具
Summary
在此, 我们提出了一个快速, 简便, 低成本的方法, 以制造定制的烷模具, 可用于生产水凝胶为基础的工程组织复杂的几何。我们还描述了机械和组织学评估的结果, 对使用这种技术生产的工程心脏组织进行了研究。
Abstract
随着组织工程领域的不断成熟, 对各种组织参数 (包括组织形态) 的兴趣也越来越大。在千分尺上操作组织形状可以直接对细胞进行排列, 改变有效的机械性能, 并解决与养分扩散有关的限制。此外, 制备组织的容器可以对组织施加机械约束, 导致应力场, 从而进一步影响细胞和基质结构。具有高度可重现性的形状组织还具有用于体外检测的实用程序, 其中样本维度至关重要, 例如整个组织机械分析。
本手稿描述了利用激光蚀刻丙烯酸制备的负主模的替代制造方法: 这些模具与烷的性能良好, 允许设计厘米刻度和特征的尺寸尺寸小于25µm, 可快速设计和制造的低成本和最低的专业知识。最低的时间和成本要求, 使激光蚀刻模具被迅速地重复, 直到一个优化的设计是确定的, 并易于适应, 以适应任何兴趣的检测, 包括那些超出组织工程领域。
Introduction
在过去的两年中, 软光刻被广泛地用作一种制造技术来支持科学研究, 特别是在微流体、材料研究和组织工程领域, 1, 2, 3。复制成型, 其中一个对象具有一个理想的形状是由负主模创建, 提供了一个方便和低成本的方法, 生产积极的, 可用于铸造成型水凝胶。但是, 所需的负主模具通常是使用昂贵、耗时、尺寸有限的微细加工技术生产的, 需要干净的房间空间和精良的设备。虽然3D 打印提供了一种潜在的替代方案, 但由于低成本打印机的分辨率限制以及常见的3D 打印机聚合物和可抑制固化的聚硅烷之间的化学作用, 它的实用性受到了一定的限制。
激光切割和蚀刻材料, 如塑料, 木材, 玻璃和金属的切割系统最近变得大大降低成本, 因此更容易为制造研究工具。商用级激光切割机能够制造厘米刻度上的物体, 其最小特性小于25µm, 而且还需要最少的训练、专门知识和使用时间。在微流体设备的制造过程中, 对其进行了激光消融, 但据我们所知, 没有手稿描述了从激光切割负主模具中可以制造毫米和厘米级模的工艺 4.
我们使用这一技术主要是为了改善营养扩散, 细胞对齐, 和机械性能5,6,7, 以控制工程组织的形状。然而, 这种技术的通用性允许在任何领域使用模压水凝胶感兴趣, 如药物输送和材料科学研究8。利用激光切割机, 可对几乎任何几何无悬垂 (这将抑制去除没有一个多部分的模具, 这是超出本手稿的范围), 并适合在激光床的尺寸。
Protocol
Representative Results
Discussion
定制的与组织培养相适应的注塑模几何图形在调整重要的工程组织性能方面有很大用处, 如细胞对准、扩散速率和有效刚度。此外, 这些模具对于为需要几何的分析应用程序准备组织非常有用, 如机械测试16,17。从激光切割负主模具中制备这些器件提供了一种快速、简便、低成本的利用这些工具的方法, 特别是与传统微细加工的时间和成本相比较。激光切?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者承认来自 NIH R00 HL115123 和布朗大学工程学院的资助。他们也感谢布朗设计车间和克里斯公牛为训练和支持与激光切割机。
Materials
| Item | |||
| Bovine fibrinogen | Sigma | F8630-5G | Constructs |
| Bovine thrombin | Sigma | T6634-250UN | Constructs |
| Bovine aprotinin | Sigma | 10820-25MG | Constructs |
| Rat tail collagen I, 4 mg/mL | Advanced Biomatrix | 5153-100MG | Constructs |
| Sodim chloride | Fisher | BP358-10 | Constructs |
| PBS | Life Technologies | 14190-250 | Constructs |
| Fine forceps | Fine Science Tools | 11252-20 | Constructs |
| Sylgard 184 silicone elastomer | Corning | 4019862 | PDMS Molds |
| Lab tape | Fisher | 15-901-5R | PDMS Molds |
| Acrylic, 1/4" thick | McMaster-Carr | 8560K356 | PDMS Molds |
| HEPES Buffer, 1 M | Sigma | H3537-100ML | Constructs |
| RPMI 1640 medium, powder | Fisher | 31800-089 | Constructs |
| Calcium chloride dihydrate | Fisher | AC423520250 | Constructs |
| Magnesium chloride hexahydrate | Fisher | M33 500 | Constructs |
| Potassium chloride | Sigma | P9541-500G | Constructs |
| Sodium phosphate dibasic heptahydrate | Sigma | S9390-500G | Constructs |
| Glucose | Sigma | G5767-25G | Constructs |
| OCT | VWR | 25608-930 | Histology |
| Frozen block molds | VWR | 25608-916 | Histology |
| Hematoxylin | Fisher | 3530 1 | Histology |
| Eosin Y | Fisher | AC152880250 | Histology |
| Fast green FCF | Fisher | AC410530250 | Histology |
| Software | |||
| Illustrator | Adobe Systems | Vector Graphics | |
| Inkscape | (Open Source) | Vector Graphics | |
| UCP (Universal Control Panel) | Universal Laser Systems | Laser Cutter Interface | |
| Equipment | |||
| PLS6.75 Laser Cutter | Universal Laser Systems | Laser Cutter | |
| Micromechanical Analyzer | Aurora Scientific | 1530A with 5 mN load cell | Mechanical Analysis |
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