携带人多能干细胞球体的核壳微胶囊的微流体制造
Summary
本文介绍了使用同轴流聚焦装置封装人多能干细胞（hPSC）。我们证明，这种微流体封装技术能够有效地形成hPSC球体。
Abstract
人类多能干细胞（hPSCs）的三维（3D）或球状培养物具有改善分化结果和可扩展性的好处。在本文中，我们描述了一种用于hPSC球体的稳健且可重复形成的策略，其中利用同轴流聚焦装置将hPSC卡入核壳微胶囊内。核心溶液含有hPSCs的单细胞悬浮液，通过掺入高分子量聚乙二醇（PEG）和密度梯度培养基而变得粘稠。壳流由PEG-4臂马来酰亚胺或PEG-4-Mal组成，并沿着核心流流流向两个连续的油结。液滴形成发生在第一个油结处，壳溶液包裹在核心周围。壳的化学交联发生在第二个油连接处，通过向这些液滴引入二硫醇交联剂（1，4-二硫代噻嗪醇或DTT）。交联剂 通过 点击化学与马来酰亚胺官能团反应，导致在微胶囊周围形成水凝胶壳。我们的封装技术以每秒10粒胶囊的速度生产400μm直径的胶囊。由此产生的胶囊具有水凝胶壳和水性核心，允许单个细胞迅速组装成聚集体并形成球状体。包封过程不会对hPSCs的生存能力产生不利影响，包封后3天观察到>95%的存活率。为了进行比较，包封在固体凝胶微粒（无水性核心）中的hPSCs在包封后3天未形成球状体，并且具有<50%的活力。包封后48 h内核壳微胶囊内hPSCs的球状体形成，球体直径与细胞接种密度成函数关系。总体而言，该协议中描述的微流体封装技术非常适合hPSCs封装和球体形成。
Introduction
由于这种培养形式1，2，3提供了改进的多能性和分化潜力，因此对人多能干细胞（hPSCs）的3D培养物有相当大的兴趣。hPSC通常通过生物反应器，微孔，水凝胶和聚合物支架4，5，6形成球体或其他3D培养形式。封装为将单个hPSC组织成球体提供了另一种方法。一旦封装了hPSC球体，就可以轻松处理并转移到微量滴定板中，以进行分化，疾病建模或药物测试实验。将hPSC包裹在水凝胶层中还可以保护细胞免受剪切损伤，并允许以高搅拌速率在生物反应器中培养球体7。
我们的干细胞封装方法随着时间的推移而发展。首先，我们专注于固体水凝胶微粒，并展示了小鼠胚胎干细胞（mESCs）的成功封装和培养8。然而，值得注意的是，当包封在这种水凝胶微粒中时，人类胚胎干细胞（hESCs）具有低活力，可能是由于这些细胞在包封后更需要重新建立细胞 – 细胞接触。我们推断，具有水性核心的异质微胶囊可能更适合于依赖于细胞 – 细胞接触的快速重建的细胞的包封。用于制造水性核/水凝胶壳微胶囊的同轴流聚焦微流体装置的概念改编自He等人，但原始方法中采用了藻酸盐，而是将基于PEG的水凝胶掺入壳中。我们首先证明了核壳微胶囊10 中原代肝细胞的成功包封和球体形成，以及最近描述的hES和iPS细胞7的包封。如图 1A所示，胶囊在流动聚焦装置中制造，其中壳和核心流动流在喷射到油相之前从一侧到另一侧过渡到同轴流。核心流含有增加溶液粘度的细胞和添加剂（非反应性PEG MW 35kD和碘克沙醇 – 商业名称OptiPrep），而壳流含有反应性分子（PEG-4-Mal）。连续的同轴流被离散化为保留核壳结构的液滴。通过暴露于二硫醇交联剂（DTT）使核壳结构永久化，DTT 通过 点击化学与PEG-4-Mal反应，并形成薄（〜10μm）的水凝胶表皮或壳。在乳液破碎并且胶囊被转移到水相之后，PEG分子从核心扩散并被水分子取代。这导致水性核心和水凝胶壳微胶囊。
下面提供了有关如何制造微流体器件，如何制备细胞以及如何进行hPSC封装的分步说明。
Protocol
Representative Results
Discussion
这里描述的封装过程导致hPSC球体的可重复形成。微胶囊形式可以很容易地将球体分配到微量滴定板的孔中，用于旨在改善/优化分化方案或测试疗法的实验。包封的干细胞球体也可用于悬浮培养物，其中水凝胶壳保护细胞免受剪切诱导的损伤7。
协议中有几个关键步骤。将核心、壳和油流的流速保持在“协议”部分所述的范围内非常重要。如果壳流速太低，?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究部分得到了梅奥诊所再生医学中心，J. W. Kieckhefer基金会，Al Nahyan基金会，明尼苏达州再生医学（RMM 101617 TR 004）和NIH（DK107255）的资助。
Materials
| 0.22 µm Syringe Filters | Genesee Scientific | 25-244 | |
| 1 ml syringe luer-lock tip | BD | 309628 | |
| 1x DPBS | Corning | 23220003 | |
| 4-arm PEG maleimide, 10kDa | Laysan Inc. | 164-68 | |
| 5 ml syringe luer-lock tip | BD | 309646 | |
| 6-WELL NON-TREATED PLATE | USA Scientific | CC7672-7506 | |
| Aquapel Applicator Pack | Aquapel Glass Treatment | 47100 | |
| CAD software | Autodesk | AutoCAD v2020 | |
| CELL STRAINER 100 µm pore size | cardinal | 335583 | |
| Chlorotrimethylsilane | Aldrich | 386529-100mL | |
| Countess II FL Automated Cell Counter | Life technology | A27974 | |
| Digital hot plate | Dataplate | ||
| Digital vortex mixer | Fisher Scientific | 215370 | |
| Distilled water | Gibco | 15230-162 | |
| Dithiotheritol (DTT) | Sigma | D0632-10G | |
| DMEM/F12 media | gibco | 11320-033 | |
| Falcon 15 mL Conical Centrifuge Tubes | Fisher scientific | 14-959-53A | |
| Fisherbrand accuSpin Micro 17 Microcentrifuge | live | 13-100-675 | |
| HERACELL VIOS 160i CO2 Incubator | Thermo Scientific | 50144906 | |
| Inverted Fluorescence Motorized Microscope | Olympus | Olympus IX83 | |
| Laurell Spin Coaters | Laurell Technologies | WS-650MZ-23NPPB | |
| Live/Dead mammalian staining kit | Fisher | L3224 | |
| Magic tape | Staples | 483535 | |
| Micro Medical Tubing (0.015" I.D. x 0.043" O.D.) | Scientific Commodities, Inc | BB31695-PE/2 | |
| Micro stir bar | Daigger Scientific | EF3288E | |
| MilliporeSigma Filter Forceps | Fisher scientific | XX6200006P | |
| Mineral oil | Sigma | M8410-1L | |
| mTeSR 1 Basal Medium | STEMCELL TECHNOLOGY | 85850 | |
| Needles-Stainless Steel 14 Gauge | CML supply | 901-14-025 | |
| Needles-Stainless Steel 15 Gauge | CML supply | 901-15-050 | |
| OptiPrep | STEMCELL TECHNOLOGY | 7820 | |
| Oven | Thermo Scientific | HERA THERM Oven | |
| Penicillin:Streptomycin (10,000 U/mL Penicillin G, 10mg/mL Streptomycin) | Gemini | 400-109 | |
| Petri Dish 150X20 Sterile Vent | Sarstedt, Inc. | 82.1184.500 | |
| Plasma Cleaning System | Yield Engineering System, Inc. | YES-G500 | |
| Pluronic F-127 | Sigma | P2443-250G | |
| Poly(ethylene glycol) 35kDa | Sigma | 94646-250G-F | |
| PrecisionGlide Needle 27G | BD | 305109 | |
| Rock inhibitor Y-27632 dihydrocloride | SELLECK CHEM | S1049-10mg | |
| Silicon wafer 100mm | University Wafer | 452 | |
| Slide glass (75mm ´ 25mm) | CardinalHealth | M6146 | |
| Span 80 | Sigma | S6760-250ML | |
| SpeedMixer | Thinky | ARE-310 | |
| Spin-X Centrifuge Tube Filter (0.22 µm) | Costar | 8160 | |
| SU-8 2025 | Kayaku Advanced Materials | Y111069 0500L1GL | |
| SU-8 developer | Kayaku Advanced Materials | Y020100 4000L1PE | |
| Surgical Design Royaltek Stainless Steel Surgical Scalpel Blades | fisher scientific | 22-079-684 | |
| SYLGARD TM 184 Silicone Elastomer Kit (PDMS) | Dow Corning | 2065622 | |
| Syringe pump | New Era Pump System, Inc | NE-4000 | |
| Triethanolamine | Sigma-aldrich | T58300-25G | |
| TrypLE Express | Gibco | 12604-013 | |
| Tygon Tubing (0.02" I.D. x 0.06" O.D.) | Cole-Parmer | 06419-01 | |
| Tygon Tubing (0.04" I.D. x 0.07" O.D.) | Cole-Parmer | 06419-04 | |
| Ultrasonic cleaner FS20D | Fisher Scientific | CPN-962-152R | |
| Vacuum desiccator | Bel-Art | F42025-0000 | |
| Zeiss Stemi DV4 Stereo Microscope 8x-32x | ZEISS | 435421-0000-000 | |
| μPG 101 laser writer | Heidelberg Instruments | HI 1128 |
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