将静态屏障组织模型转化为动态微生理系统
Summary
该协议描述了一种可重新配置的基于膜的细胞培养平台，该平台将开孔形式与流体流动功能集成在一起。该平台与标准方案兼容，并允许在开孔和微流控培养模式之间进行可逆过渡，以满足工程和生物科学实验室的需求。
Abstract
微生理系统是小型化的细胞培养平台，用于在实验室环境中模拟人体组织的结构和功能。然而，这些平台尚未在生物科学实验室中得到广泛采用，尽管缺乏流体流动能力，但基于膜的开放式方法仍是模拟组织屏障的黄金标准。这个问题主要归因于现有的微生理系统与为明井系统开发的标准协议和工具不兼容。
在这里，我们提出了一种协议，用于创建具有开井结构、流量增强能力以及与传统协议兼容的可重构膜平台。该系统采用磁性组装方法，可在开井和微流体模式之间进行可逆切换。通过这种方法，用户可以灵活地使用标准方案以开孔形式开始实验，并根据需要添加或删除流动功能。为了证明该系统的实际用途及其与标准技术的兼容性，以开孔形式建立了内皮细胞单层。该系统被重新配置以引入流体流动，然后切换到开孔形式以进行免疫染色和 RNA 提取。由于其与传统开井方案的兼容性和流量增强能力，这种可重构设计有望被工程和生物科学实验室采用。
Introduction
血管屏障是将血室与周围组织分开的关键界面。它们通过吸引免疫细胞、控制分子通透性和屏蔽病原体侵入组织，在维持体内平衡方面发挥着关键作用 1,2。已经开发了体外培养模型来模拟体内微环境，从而能够系统地研究影响健康和疾病状态下屏障特性的因素和条件3,4。
对于这种培养模型，最广泛使用的方法是类似Transwell的“开孔”配置5，其中多孔的、迹状蚀刻的培养膜将充满培养基的隔室分开（图1A）。在这种形式下，细胞可以接种在膜的任一侧，并且已经开发了广泛的实验方案。然而，这些系统提供支持屏障成熟和模拟体内免疫细胞循环所必需的流体流动的能力有限 5,6。因此，它们不能用于需要引入药物剂量、机械刺激或流体诱导剪切应力的动态流动的研究 6,7,8。
为了克服开井系统的局限性，已经开发了将多孔培养膜与可单独寻址的流体通道相结合的微流体平台9。这些平台可精确控制流体路线、灌注和化合物的引入、受控剪切刺激和动态细胞添加功能7,10,11,12,13。尽管微流控平台提供了先进的功能，但由于复杂的微流控方案及其与既定的实验工作流程不兼容，它们尚未在生物科学实验室中得到广泛采用4,10,14。
为了弥合这些技术之间的差距，我们提出了一种协议，该协议采用磁性可重构的基于模块的系统。该系统可以根据实验的具体需要在开孔和微流控模式之间轻松切换。该平台具有一个开孔装置，称为m-μSiM（由硅膜实现的模块化微生理系统），具有100 nm厚的培养膜（纳米膜）。该纳米膜具有高孔隙率（15%）和玻璃状透明度，如图1B所示。它在物理上将顶部隔室与底部通道分开，允许跨生理长度尺度 15 的分子运输。与传统的轨迹蚀刻膜不同，传统的轨迹蚀刻膜在用明场成像对活细胞进行成像方面存在已知的挑战，纳米膜具有良好的光学和物理特性，可以清晰地看到膜表面两侧的细胞15,16,17。
本协议概述了专用播种和流动模块的制造，并解释了平台的磁性重构。它演示了如何在静态和动态条件下利用该平台建立内皮屏障。该演示表明，内皮细胞沿流动方向排列，在剪切刺激下剪切敏感基因靶标上调。
Protocol
该设计可以根据实验要求和最终用户的偏好在各种模式下使用。在每次实验之前，请查阅 图2 所示的决策流程图，以确定方案的必要步骤和模块。例如，如果用户打算在整个实验过程中保持开孔格式，以直接将其与Transwell型系统进行比较，则细胞接种不需要图案模板。核心模块是市售的（见 材料表），超薄纳米膜可以从具有不同孔隙率和孔径的材料库中进行选?…
Representative Results
如图6A所示，开井岩心模块最初位于由下部外壳和盖玻片形成的特定空腔内。随后，将包括微通道和接入端口的流量模块插入核心模块的孔中。如图6B所示，由于嵌入在下部和上部外壳中的磁体之间的磁吸引力，流量模块牢固地密封在膜的硅支撑层上。为了评估这种磁闭锁机制的有效性，进行了爆破压力测试，证明该系统可以承受高达 38.8 ± 2.4 kPa 的死?…
Discussion
该协议的目的是开发一种实用的方法，将流动能力整合到具有超薄纳米膜的开孔平台中。在这种设计中，采用了磁闭锁方法，允许在实验期间在开井和流体模式之间切换，并结合了两种方法的优点。与传统的永久粘合平台不同，磁性锁存允许在实验工作流程16,25,26,27的方便点拆卸平台。在这个平台中?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究部分由美国国立卫生研究院资助，奖励编号为R43GM137651、R61HL154249、R16GM146687和NSF授予CBET 2150798。作者感谢RIT机械车间的铝模具制造。内容完全由作者负责，并不一定代表美国国立卫生研究院的官方观点。
Materials
| 0.5 x 0.86 Micro Flow tubes | Langer Instruments | WX10-14 & DG Series | |
| 1 mm Disposable Biopsy Punches, Integra Miltex | VWR | 95039-090 | |
| 1x PBS 7.4 pH | ThermoFisher Scientific | 10010023 | |
| 20 GAUGE IT SERIES DISPENSING TIP | Jensen Global | JG20-1.5X | |
| 21 GAUGE NT PREMIUM SERIES ANGLED DISPENSING TIP | Jensen Global | JG21-1.0HPX-90 | |
| 3M 467 MP Pressure senstitive adhesive (PSA) | DigiKey | 3M9726-ND | |
| 3M 468 MP Pressure senstitive adhesive (PSA) | DigiKey | 3M9720-ND | |
| AlexaFluor 488 conjugated phalloidin | ThermoFisher Scientific | A12379 | |
| Applied Biosystems TaqMan Fast Advanced Master Mix | Thermo Fisher Scientific | 4444556 | |
| Bovine Serum Albumin (BSA), Fraction V, 98%, Reagent grade, Alfa Aesar, Size = 10 g | VWR | AAJ64100-09 | |
| Clear Scratch- and UV-Resistant Cast Acrylic Sheet | McMaster-Carr | 8560K171 | 12" x 12" x 1/16" |
| Clear Scratch- and UV-Resistant Cast Acrylic Sheet | McMaster-Carr | 8589K31 | 12" x 12" x 3/32" |
| Clear Scratch- and UV-Resistant Cast Acrylic Sheet | McMaster-Carr | 8560K191 | 12" x 12" x 7.64" |
| Corning Fibronectin, Human, 1 mg | Corning | 47743-728 | |
| Cover Glasses, Globe Scientific, L x W = 24 x 60 mm | VWR | 10118-677 | |
| DOW SYLGARD 184 SILICONE ENCAPSULANT CLEAR 0.5 KG KIT | Ellsworth Adhesives | 4019862 | |
| EGM-2 Endothelial Cell Growth Medium-2 BulletKit | Lonza | CC-3162 | |
| Fixture A1&A2 | SiMPore Inc. | NA | |
| Fixture B1&B2 | SiMPore Inc. | NA | |
| High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor | Thermo Fisher Scientific | 4374966 | |
| Human umbilical vein endothelial cells (HUVEC) | ThermoFisher Scientific | C0035C | |
| LIVE/DEAD Cell Imaging Kit (488/570) | Thermo Fisher Scientific | R37601 | |
| Molecular Probes Hoechst 33342, Trihydrochloride, Trihydrate | Thermo Fisher Scientific | H3570 | |
| Nickel-plated magnets (4.75 mm diameter, 0.34 kg pull force) | K&J Magnetics | D31 | 3/16" dia. x 1/16" thick |
| Paraformaldehyde, 4% w/v aq. soln., methanol free, Alfa Aesar | Fisher Scientific | aa47392-9M | |
| Peristaltic Pump | Langer Instruments | BQ50-1J-A | |
| Photoresist SU-8 developer solution | Fisher Scientific | NC9901158 | |
| PVDF syringe filters | PerkinElmer | 2542913 | |
| Silicon wafer | University wafer,USA | 1196 | |
| SU-8 3050 | Fisher Scientific | NC0702369 | |
| Target gene: eNOS (Hs01574659_m1) | ThermoFisher Scientific | 4331182 | |
| Target gene: GAPDH (Hs02786624_g1) | ThermoFisher Scientific | 4331182 | |
| Target gene: KLF2 (Hs00360439_g1) | ThermoFisher Scientific | 4331182 | |
| Thermo Scientific Pierce 20x PBS Tween 20 | Thermo Fisher Scientific | 28352 | |
| Transport Tube Sample White caps, 5 mL, Sterile | VWR | 100500-422 | |
| TRI-reagent | ThermoFisher Scientific | AM9738 | |
| Ultrathin Nanoporous Membrane Chip | SiMPore Inc. | NPSN100-1L | The design is compatible with all of SiMPore membranes |
| uSiM component 1 | SiMPore Inc. | NA | |
| uSiM component 2 | SiMPore Inc. | NA |
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Citar este artigo
Mansouri, M., Hughes, A. R., Audi, L. A., Carter, A. E., Vidas, J. A., McGrath, J. L., Abhyankar, V. V. Transforming Static Barrier Tissue Models into Dynamic Microphysiological Systems. J. Vis. Exp. (204), e66090, doi:10.3791/66090 (2024).