牵引显微镜与微流体集成，用于化学集体迁移
Summary
发育、伤口愈合和癌症转移中的集体细胞迁移通常受生长因子或信号分子梯度的引导。本文介绍了一个实验系统，将牵引显微镜与微流体系统相结合，并演示了如何在生物化学梯度下量化集体迁移的力学。
Abstract
细胞改变迁移模式以响应化学刺激，包括刺激的梯度。细胞向化学梯度方向迁移，称为化学梯度，在发育、免疫反应、伤口愈合和癌症转移中起着重要作用。虽然化学疗法调节单个细胞的迁移以及体内细胞的集合，体外研究侧重于单细胞化疗，部分原因是缺乏适当的实验工具。为了填补这一空白，这里描述的是一个独特的实验系统，它结合了微流体和微模式，以展示化学梯度对集体细胞迁移的影响。此外，牵引显微镜和单层应力显微镜被纳入系统，以表征基基上以及相邻细胞之间的细胞力变化。作为概念证明，在肝细胞生长因子（HGF）的梯度下测试马丁-达比（MDCK）细胞微模式圆形岛屿的迁移。这是一种已知的散射因子。研究发现，位于HGF浓度较高的细胞比细胞岛内另一侧的细胞迁移得更快。在同一岛内，两侧的细胞牵引力相似，但高HGF浓度的一侧细胞间应力要低得多。这一新型实验系统为研究细胞群化学迁移机制提供了新的机会。
Introduction
细胞迁移在生物系统中是一个基本现象，涉及组织形成，免疫反应，伤口愈合1，2，3。细胞迁移也是癌症等某些疾病的重要过程。细胞通常作为一个组而不是单个迁移，这称为集体细胞迁移 4，5。对于细胞集体移动，对微环境的感知至关重要6。例如，细胞感知物理化学刺激，并通过改变运动性、细胞-基质相互作用和细胞-细胞相互作用来做出反应，从而沿着化学梯度7、8发生定向迁移。 9，10.基于这种联系，在片上实验室技术方面取得了迅速的进步，这些技术可以创造出控制良好的化学微环境，如化学吸引剂11、12、13的梯度。.虽然这些基于芯片的实验室微流体学以前曾用于研究细胞团或细胞球体的化学细胞14、15、16、17，但它们主要用于单细胞迁移18，19，20，21。细胞集体对化学梯度反应的机制仍然不太了解14，22，23，24，25，26.因此，开发一个能够对可溶性因子进行时空控制以及对细胞生物物理进行原地观察的平台将有助于解开集体细胞迁移背后的机制。
这里开发和描述的是一个多通道微流体系统，能够产生可溶性因子的浓度梯度，调节模式细胞簇的迁移。在这项研究中，选择肝细胞生长因子（HGF）来调节马丁-达比管肾（MDCK）细胞的迁移行为。HGF已知能衰减细胞完整性，增强细胞的活力27，28。在微流体系统中，傅立叶转换牵引显微镜和单层应力显微镜也进行了整合，从而可以分析组成细胞在响应HGF时引起的运动力、收缩力和细胞间张力梯度。在同一岛内，位于HGF浓度较高的细胞迁移速度更快，细胞间应激水平低于HGF浓度较低的细胞侧。结果表明，这一新的实验系统适用于在各种可溶性因子化学梯度下，在涉及集体细胞迁移的研究领域的其他问题。
Protocol
注：SU-8模具的平版印刷模具（厚度 = 250 μm）和微通道零件（厚度 = 150 μm）、玻璃蚀刻（深度 = 100 μm）和铸件制造均通过使用计算机辅助设计软件向制造商发送设计进行外包。 1. 聚二甲基硅氧烷（PDMS）模具和微通道的制造 设计模具和微通道的微模式。 在硅晶片（直径 4 英寸）上制造或外包 SU-8 模具（模具厚度为 ±250 μm，微通道为 ±150 μm）。 将基弹?…
Representative Results
为了探索化学梯度下的集体迁移，将微流体系统与牵引显微镜集成在一起（图1）。为了构建集成系统，聚丙烯酰胺 （PA） 凝胶被浇在定制切割的玻璃上，MDCK 细胞在由 PDMS 模具制成的微图案岛屿中播种。在本实验中，创建了12个MDCK细胞孤岛（四行乘三列，直径为+700μm）。在附着在PA凝胶上的细胞后，PDMS模具被移除以启动集体迁移。预制造的微流体通道被放置在PA凝胶的顶部…
Discussion
组成细胞的集体迁移是发育和再生过程中的一个重要过程，迁移方向往往受生长因子4、23的化学梯度的引导。在集体迁移期间，细胞与相邻细胞和底层基质继续相互作用。这种机械相互作用产生一些紧急现象，如杜罗塔克42、plithot轴33和基轴43。然而，这项研究是使用单一浓度的生长因子进行的，如?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了韩国政府资助的韩国国家研究基金会（NRF）赠款的支持（No.NRF-2017R1E1A1A01075103），韩国大学助学金和BK 21 Plus计划。它还得到了国家卫生研究院（U01CA202123、PO1HL120839、T32HL007118、R01EY019696）的支持。
Materials
| 0.25% trypsin-EDTA (1X) | Gibco | 25200-056 | |
| 1 M HEPES buffer solution | Gibco | 15630-056 | |
| 1 mm Biopsy punch | Integra Miltex | 33-31AA-P/25 | |
| 100 mm petri dishes | SPL | 10100 | 100 mm diameter, 15 mm height |
| 14 mm hollow punch | ILJIN | 124-0571 | |
| 18 mm Ø Coverslip | Marienfeld-Superior | 111580 | Circular 18 mm, thickness No. 1 (0.13 to 0.16 mm) |
| 2% bis-acrylamide solution | Bio-Rad | 1610142 | Wear protective gloves, clothing, and eye protection. |
| 3-(Trimethoxysilyl)propyl methacrylate (TMSPMA) | Sigma-Aldrich | 440159-500ML | |
| 3-way stopcock | Hyupsung | HS-T-61N | CAUTION: do not use if previously opened. do not resterlize or resuse |
| 30 cm minimum volume line (for pediatric) | Hyupsung | HS-MV-30 | CAUTION: do not use if previously opened. do not resterlize or resuse |
| 35 mm cell culture dish | Corning | 430165 | |
| 40% Acrylamide Solution | Bio-Rad | 1610140 | Wear protective gloves, clothing, and eye protection. |
| 75 cm minimum volume line (for pediatric) | Hyupsung | HS-MV-75 | CAUTION: do not use if previously opened. do not resterlize or resuse |
| acetic acid | J.T. Baker | JT9508-03 | |
| Ammonium persulfate (APS) | Bio-Rad | 1610700 | |
| Antibiotic-Antimycotic | Gibco | 15240-062 | |
| Bottom glass chip | MicroFIT | 24 x 24 x 1 mm, custom-made, rectangular groove (6 x 12 mm, depth : 100 μm) | |
| Collagen typeI, Rat tail | Corning | 354236 | |
| Custom glass holder | Han-Gug Mechatronics | custom-made | |
| Dulbecco's Modified Eagle's Medium (DMEM) | Welgene | LM 001-11 | |
| Dulbecco's Phosphate Buffered Saline (PBS) | Biowest | L0615-500 | w/o Magnesium, Calcium |
| Fetal bovine serum (FBS) | Gibco | 26140-179 | |
| FluoSpheres amine-modified microspheres | Invitrogen | F8764 | 0.2 µm, yellow-green fluorescent(505/515) |
| Hepatocyte Growth Factor (HGF) | Sigma-Aldrich | H1404-5UG | recombinant, human |
| JuLI stage live cell imaging system | NanoEnTek In | Automated X-Y-Z stage and fluorsent imaging Incubator-compatible (37 °C and 5% CO2) | |
| Madin-Darby Canine Kidney (MDCK) cell | type II | ||
| Oxygen plasma system | Femto Science | CUTE-MPR | |
| Pluronic F-127 | Sigma-Aldrich | P2443-250G | |
| Rhodamine B isothiocyanate–dextran | Sigma-Aldrich | R9379-100MG | 70 kDa, used to estimate spatiotemporal distribution of HGF in the microfluidic channel |
| Steril hypodermic needle 18 G | KOVAX | Trim the tip of the needle and bend it 90 degrees for connecting in/out ports with volume line | |
| Sticky tape | 3M/Scotch | 810D | 33 m x 19 mm |
| SU-8 master molds | MicroFIT | 4” diameter, custom-made | |
| sulfosuccinimidyl 6-(4’-azido-2’-nitrophenylamino)hexanoate (Sulfo-SANPAH) | Thermo Scientific | 22589 | Store at -20°C. Store protected from moisture and light. |
| Sylgard 184 Elastomer Kit | Dow Corning | PDMS | |
| Syringe pump | Chemyx Inc. | model fusion 720 | withdraw fluid |
| Syringes | KOVAX | 1, 3, 5, 10, or 50 cc for using inlet reservoir or outlet syringe pump | |
| tetramethylethylenediamine (TEMED) | Bio-Rad | 1610800 | Wear protective gloves, clothing, and eye protection. |
| Ultraviolet (UV) lamp | UVP LLC | 95-0248-02 | 365 nm wavelength |
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Cite This Article
Jang, H., Kim, J., Shin, J. H., Fredberg, J. J., Park, C. Y., Park, Y. Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration. J. Vis. Exp. (152), e60415, doi:10.3791/60415 (2019).