具有长距离纤维排列的微工程3D胶原蛋白水凝胶
Summary
该协议演示了使用沿流体流动方向改变几何形状的微流体通道来产生拉伸应变（拉伸）以在3D胶原水凝胶（厚度<250μm）中对齐纤维。由此产生的对齐延伸几毫米，并受拉伸应变率的影响。
Abstract
排列的胶原I（COL1）纤维引导肿瘤细胞运动，影响内皮细胞形态，控制干细胞分化，是心脏和肌肉骨骼组织的标志。为了研究细胞对 体外排列微环境的反应，已经开发了几种协议来生成具有定义纤维排列的COL1基质，包括磁性，机械，基于细胞和微流体方法。其中，微流体方法提供了先进的功能，例如精确控制流体流动和细胞微环境。然而，用于高级 体外 培养平台生成对齐COL1基质的微流体方法仅限于COL1纤维的薄“垫”（厚度<40μm），其延伸距离小于500μm，不利于3D细胞培养应用。在这里，我们提出了一种协议来制造3D COL1矩阵（厚度为130-250μm），该矩阵具有微流体装置中定义的纤维排列的毫米级区域。该平台提供先进的细胞培养功能，通过直接访问用于细胞培养的微工程基质来模拟结构化组织微环境。
Introduction
细胞驻留在称为细胞外基质（ECM）的复杂3D纤维网络中，其中大部分由结构蛋白I型胶原蛋白（COL1）组成1，2。ECM的生物物理特性为细胞提供指导线索，作为回应，细胞重塑ECM微结构3，4，5。这些相互的细胞-基质相互作用可以产生排列的COL1纤维结构域6，其促进肿瘤环境中的血管生成和细胞侵袭7，8，9并影响细胞形态10，11，12，极化13和分化14。排列的胶原纤维还可以促进伤口愈合15，在组织发育中起关键作用16，并有助于长距离细胞通讯17，18。因此，在体外复制天然COL1纤维微结构是开发结构化模型以研究细胞对对齐微环境的反应的重要一步。
微流控细胞培养系统已被确立为开发微生理系统（MPS）的首选技术19，20，21，22，23。利用有利的微尺度缩放效应，这些系统提供对流体流动的精确控制，支持机械力的受控引入，并定义微通道21，24，25，26，27内的生化微环境。MPS平台已被用于模拟组织特异性微环境并研究多器官相互作用28。同时，水凝胶已被广泛探索，以概括在体内观察到的ECM的3D力学和生物学影响29，30。 随着越来越重视将3D培养与微流体平台相结合，许多方法可以在微流体装置中结合COL1水凝胶31，32，33。然而，在微流体通道中对齐COL1水凝胶的方法仅限于<1mm宽的通道中的薄2D“垫”（厚度<40μm），在对齐的3D微环境中模拟细胞反应的潜力有限31，34，35，36。
为了在微流体系统中实现对齐的3D COL1水凝胶，已经表明，当自组装COL1溶液暴露于局部拉伸流（沿流向的速度变化）时，所得COL1水凝胶显示出与它们经历的拉伸应变率的大小成正比的纤维取向程度37，38.该协议中的微通道设计在两个方面是独一无二的;首先，分段设计将局部拉伸应变引入COL1解决方案，其次，其“两件式”结构允许用户对齐COL1光纤，然后拆卸通道以开放格式直接访问对齐的光纤。这种方法可以进一步用于开发模块化微流体平台，开发具有有序COL1矩阵的微生理系统。以下协议描述了制造分段微通道的过程，并详细介绍了使用这些通道来对齐牛 atelo COL1。该协议还提供了以开放孔形式在COL1上培养细胞的说明，并讨论了使用模块化磁性基础层向平台添加功能。
Protocol
1. 两件式通道和模块化平台底座的制造 注意：微流体通道由两部分构成 – 微流体通道“切口”，它是从规定厚度的聚二甲基硅氧烷（PDMS）片上切割的剃刀，以及通道盖，可逆地粘合到切口并形成通道。通道被聚甲基丙烯酸甲酯（PMMA）框架包围，该框架将充当介质储液器（图1）。PMMA框架还可用于磁性锁存专用模块以增加功能。 PDMS ?…
Representative Results
当自组装COL1解流过横截面积减小的通道时，COL1解的流速（v x）沿两段之间的收缩长度（∂x）局部增加一个量级∂v x，导致拉伸应变率（ε̇），其中ε̇ = ∂v x/∂x。拉伸应变率可以通过流体速度计算，流体速度是使用粒子图像测速法（PIV）测量的，如图2所示。 先前，已经表明局部拉伸应变促进长距离COL1纤维排列37…
Discussion
已经使用磁性方法、机械应变的直接应用和微流体技术描述了生成具有排列纤维的 COL1 矩阵的协议47。微流体方法通常用于创建微生理系统，因为它们具有明确定义的流动和运输特性，可以精确控制生化微环境。由于排列的COL1纤维在病理生理过程中（如伤口愈合，肿瘤细胞侵袭和组织发育）提供了关键的指导性线索，因此在微流体系统中生成排列的COL1基质是朝着开发生物学相关…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院的部分支持，奖励号为R21GM143658，美国国家科学基金会的资助号为2150798。内容完全由作者负责，不一定代表资助机构的官方观点。
Materials
| (3-Aminopropyl)triethoxysilane, 99% (APTES) | Sigma Aldrich | 440140-100ML | |
| 20 Gauge IT Series Angled Dispensing Tip | Jensen Global | JG-20-1.0-90 | |
| 3/16" dia. x 1/16" thick Nickel Plated Magnet | KJ Magnetics | D31 | |
| 3M (TC) 12X12-6-467MP | DigiKey | 3M9726-ND | |
| ACETONE ACS REAGENT ≥99.5% | Signa Aldrich | 179124-4L | |
| BD-20AC LABORATORY CORONA TREATER | Electro-Technic Products | 12051A | |
| Bovine Serum Albumin (BSA), Fraction V, 98%, Reagent Grade, Alfa Aesar | VWR | AAJ64100-09 | |
| Clear cast acrylic sheet | McMaster-Carr | 8560K181 | |
| Corning 100 mL Trypsin 10x, 2.5% Trypsin in HBSS [-] calcium, magnesium, phenol red, Porcine Parvovirus Tested | VWR | 45000-666 | |
| Countess II Automated Cell Counter | Thermo Fisher Scientific | AMQAX1000 | |
| CT-FIRE software | LOCI – University of Wisconsin | ||
| EGM-2 Endothelial Cell Growth Medium-2 BulletKit, (CC-3156 & CC-4176), Lonza CC-3162, 500 mL | Lonza | CC-3162 | |
| Glutaraldehyde 50% in aqueous solution, Reagent Grade, Packaging=HDPE Bottle, Size=100 mL | VWR | VWRV0875-100ML | |
| Graphtec CELITE-50 | Graphtec | CE LITE-50 | |
| HEPES (1 M) | Thermo Fisher Scientific | 15-630-080 | |
| High-Purity Silicone Rubber .010" Thick, 6" X 8" Sheet, 55A Durometer | McMaster-Carr | 87315K62 | |
| Human Umbilical Vein Endothelial cells | Thermo Fisher Scientific | C0035C | |
| Invitrogen Trypan Blue Stain (0.4%) | Thermo Fisher Scientific | T10282 | |
| Isopropanol | Fisher Scientific | A4154 | |
| Laser cutter | Full Spectrum | 20×12 H-series | |
| Microfluidics Syringe pump | New Era Syringe Pumps | NE-1002X | |
| Microman E Single Channel Pipettor, Gilson, Model M1000E | Gilson | FD10006 | |
| Molecular Probes Alexa Fluor 488 Phalloidin | Thermo Fisher Scientific | A12379 | |
| Molecular Probes Hoechst 33342, Trihydrochloride, Trihydrate | Thermo Fisher Scientific | H3570 | |
| Nutragen Bovine Atelo Collagen | Advanced BioMatrix | 5010-50ML | |
| Pbs (10x), pH 7.4 | VWR | 70011044.00 | |
| PBS pH 7.4 | Thermo Fisher Scientific | 10010049.00 | |
| Phosphate-buffered saline (PBS, 10x), with Triton X-100 | Alfa Aesar | J63521 | |
| Replacement carrier sheet for graphtec craft ROBO CC330L-20 | USCUTTER | GRPCARSHTN | |
| Restek Norm-Ject Plastic Syringe 1 mL Luer Slip | Restek | 22766.00 | |
| Silicon wafer | University wafer | 452 | |
| Sodium Hydroxide, ACS, Packaging=Poly Bottle, Size=500 g | VWR | BDH9292-500G | |
| Sylgard 184 | VWR | 102092-312 | |
| Thermo Scientific Pierce 20x PBS Tween 20 | Thermo Fisher Scientific | 28352.00 |
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Cite This Article
Ahmed, A., Joshi, I. M., Goulet, M. R., Vidas, J. A., Byerley, A. M., Mansouri, M., Day, S. W., Abhyankar, V. V. Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment. J. Vis. Exp. (187), e64457, doi:10.3791/64457 (2022).