Name: microfluidic fabrication of core-shell microcapsules carrying human pluripotent stem cell spheroids
Uploaded: 2021-10-13T14:30:00
Description: Watch this Scientific Journal Video about Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids at JoVE.com

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。

1. 设备制造
<ol><li>使用CAD软件10，11进行微囊化装置和解离装置的设计。</li>
	<li>在硅晶圆上依次纺涂三层SU-8光刻胶（图2A），以获得具有所需高度的结构：60，100和150μm。 注：顶部和底部模具的工艺相同。<ol>
<li>用SU-8 2025负光刻胶以1，100 rpm的速度旋转涂覆干净的10厘米硅片，以产生第一个60μm层。在热板上在65°C下软烘烤3分钟...

<ol>
	<li>Zhu, Z., Huangfu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pluripotent+stem+cells:+an+emerging+model+in+developmental+biology.">Human pluripotent stem cells: an emerging model in developmental biology.</a> Development. 140 (4), 705-717 (2013).</li><li>Liu, G., David, B. T., Trawczynski, M., Fessler, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+pluripotent+stem+cells:+history,+mechanisms,+technologies,+and+applications.">Advances in pluripotent stem cells: history, mechanisms, technologies, and applications.</a> Stem Cell Reviews and Reports. 16 (1), 3-32 (2020).</li><li>Chan, S. W., Rizwan, M., Yim, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+methods+for+enhancing+pluripotent+stem+cell+expansion.">Emerging methods for enhancing pluripotent stem cell expansion.</a> Frontiers in Cell and Developmental Biology. 8, 70 (2020).</li><li>Lei, Y., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fully+defined+and+scalable+3D+culture+system+for+human+pluripotent+stem+cell+expansion+and+differentiation.">A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (52), 5039-5048 (2013).</li><li>Olmer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suspension+culture+of+human+pluripotent+stem+cells+in+controlled,+stirred+bioreactors.">Suspension culture of human pluripotent stem cells in controlled, stirred bioreactors.</a> Tissue Engineering Part C: Methods. 18 (10), 772-784 (2012).</li><li>Kraehenbuehl, T. P., Langer, R., Ferreira, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+biomaterials+for+the+study+of+human+pluripotent+stem+cells.">Three-dimensional biomaterials for the study of human pluripotent stem cells.</a> Nature Methods. 8 (9), 731-736 (2011).</li><li>Fattahi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Core-shell+hydrogel+microcapsules+enable+formation+of+human+pluripotent+stem+cell+spheroids+and+their+cultivation+in+a+stirred+bioreactor.">Core-shell hydrogel microcapsules enable formation of human pluripotent stem cell spheroids and their cultivation in a stirred bioreactor.</a> Scientific Reports. 11 (1), 1-13 (2021).</li><li>Siltanen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+fabrication+of+bioactive+microgels+for+rapid+formation+and+enhanced+differentiation+of+stem+cell+spheroids.">Microfluidic fabrication of bioactive microgels for rapid formation and enhanced differentiation of stem cell spheroids.</a> Acta Biomaterialia. 34, 125-132 (2016).</li><li>Agarwal, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+microfluidic+generation+of+pre-hatching+embryo-like+core-shell+microcapsules+for+miniaturized+3D+culture+of+pluripotent+stem+cells.">One-step microfluidic generation of pre-hatching embryo-like core-shell microcapsules for miniaturized 3D culture of pluripotent stem cells.</a> Lab on a Chip. 13 (23), 4525-4533 (2013).</li><li>Siltanen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+step+fabrication+of+hydrogel+microcapsules+with+hollow+core+for+assembly+and+cultivation+of+hepatocyte+spheroids.">One step fabrication of hydrogel microcapsules with hollow core for assembly and cultivation of hepatocyte spheroids.</a> Acta Biomaterialia. 50, 428-436 (2017).</li><li>Rahimian, A., Siltanen, C., Feyzizarnagh, H., Escalante, P., Revzin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microencapsulated+immunoassays+for+detection+of+cytokines+in+human+blood.">Microencapsulated immunoassays for detection of cytokines in human blood.</a> ACS Sensors. 4 (3), 578-585 (2019).</li><li>Kim, M., Lee, J., Jones, C. N., Revzin, A., Tae, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin-based+hydrogel+as+a+matrix+for+encapsulation+and+cultivation+of+primary+hepatocytes.">Heparin-based hydrogel as a matrix for encapsulation and cultivation of primary hepatocytes.</a> Biomaterials. 31, 3596-3603 (2010).</li><li>Shin, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodegradable+hydrogels+for+capture,+detection,+and+release+of+live+cells.">Photodegradable hydrogels for capture, detection, and release of live cells.</a> Angewandte Chemie International Edition. , (2014).</li><li>You, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactive+photodegradable+hydrogel+for+cultivation+and+retrieval+of+embryonic+stem+cells.">Bioactive photodegradable hydrogel for cultivation and retrieval of embryonic stem cells.</a> Advanced Functional Materials. , (2015).</li></ol>

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，?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m Syringe Filters</td>
			<td>Genesee Scientific</td>
			<td>25-244</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe luer-lock tip</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>1x DPBS</td>
			<td>Corning</td>
			<td>23220003</td>
			<td></td>
		</tr>
		<tr>
			<td>4-arm PEG maleimide, 10kDa</td>
			<td>Laysan Inc.</td>
			<td>164-68</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml syringe luer-lock tip</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>6-WELL NON-TREATED PLATE</td>
			<td>USA Scientific</td>
			<td>CC7672-7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquapel Applicator Pack</td>
			<td>Aquapel Glass Treatment</td>
			<td>47100</td>
			<td></td>
		</tr>
		<tr>
			<td>CAD software</td>
			<td>Autodesk</td>
			<td>AutoCAD v2020</td>
			<td></td>
		</tr>
		<tr>
			<td>CELL STRAINER 100 &#181;m pore size</td>
			<td>cardinal</td>
			<td>335583</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorotrimethylsilane</td>
			<td>Aldrich</td>
			<td>386529-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>Life technology</td>
			<td>A27974</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital hot plate</td>
			<td>Dataplate</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital vortex mixer</td>
			<td>Fisher Scientific</td>
			<td>215370</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>Gibco</td>
			<td>15230-162</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiotheritol (DTT)</td>
			<td>Sigma</td>
			<td>D0632-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 media</td>
			<td>gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 mL Conical Centrifuge Tubes</td>
			<td>Fisher scientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand accuSpin Micro 17 Microcentrifuge</td>
			<td>live</td>
			<td>13-100-675</td>
			<td></td>
		</tr>
		<tr>
			<td>HERACELL VIOS 160i CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>50144906</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Motorized Microscope</td>
			<td>Olympus</td>
			<td>Olympus IX83</td>
			<td></td>
		</tr>
		<tr>
			<td>Laurell Spin Coaters</td>
			<td>Laurell Technologies</td>
			<td>WS-650MZ-23NPPB</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead mammalian staining kit</td>
			<td>Fisher</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Magic tape</td>
			<td>Staples</td>
			<td>483535</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Medical Tubing (0.015&#34; I.D. x 0.043&#34; O.D.)</td>
			<td>Scientific Commodities, Inc</td>
			<td>BB31695-PE/2</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro stir bar</td>
			<td>Daigger Scientific</td>
			<td>EF3288E</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliporeSigma Filter Forceps</td>
			<td>Fisher scientific</td>
			<td>XX6200006P</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M8410-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR 1 Basal Medium</td>
			<td>STEMCELL TECHNOLOGY</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles-Stainless Steel&#160; 14 Gauge</td>
			<td>CML supply</td>
			<td>901-14-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles-Stainless Steel&#160; 15 Gauge</td>
			<td>CML supply</td>
			<td>901-15-050</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep</td>
			<td>STEMCELL TECHNOLOGY</td>
			<td>7820</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Thermo Scientific</td>
			<td>HERA THERM Oven</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin:Streptomycin (10,000 U/mL Penicillin G, 10mg/mL Streptomycin)</td>
			<td>Gemini</td>
			<td>400-109</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish 150X20 Sterile Vent</td>
			<td>Sarstedt, Inc.</td>
			<td>82.1184.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Cleaning System</td>
			<td>Yield Engineering System, Inc.</td>
			<td>YES-G500</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma</td>
			<td>P2443-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol) 35kDa</td>
			<td>Sigma</td>
			<td>94646-250G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlide Needle 27G</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>Rock inhibitor Y-27632 dihydrocloride</td>
			<td>SELLECK CHEM</td>
			<td>S1049-10mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer 100mm</td>
			<td>University Wafer</td>
			<td>452</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide glass (75mm &#180; 25mm)</td>
			<td>CardinalHealth</td>
			<td>M6146</td>
			<td></td>
		</tr>
		<tr>
			<td>Span 80</td>
			<td>Sigma</td>
			<td>S6760-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedMixer</td>
			<td>Thinky</td>
			<td>ARE-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin-X Centrifuge Tube Filter (0.22 &#181;m)</td>
			<td>Costar</td>
			<td>8160</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2025</td>
			<td>Kayaku Advanced Materials</td>
			<td>Y111069 0500L1GL</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 developer</td>
			<td>Kayaku Advanced Materials</td>
			<td>Y020100 4000L1PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Design Royaltek Stainless Steel Surgical Scalpel Blades</td>
			<td>fisher scientific</td>
			<td>22-079-684</td>
			<td></td>
		</tr>
		<tr>
			<td>SYLGARD TM 184 Silicone Elastomer Kit (PDMS)</td>
			<td>Dow Corning</td>
			<td>2065622</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>New Era Pump System, Inc</td>
			<td>NE-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethanolamine</td>
			<td>Sigma-aldrich</td>
			<td>T58300-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Gibco</td>
			<td>12604-013</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon Tubing (0.02&#34; I.D. x 0.06&#34; O.D.)</td>
			<td>Cole-Parmer</td>
			<td>06419-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon Tubing (0.04&#34; I.D. x 0.07&#34; O.D.)</td>
			<td>Cole-Parmer</td>
			<td>06419-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner FS20D</td>
			<td>Fisher Scientific</td>
			<td>CPN-962-152R</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum desiccator</td>
			<td>Bel-Art</td>
			<td>F42025-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Stemi DV4 Stereo Microscope 8x-32x</td>
			<td>ZEISS</td>
			<td>435421-0000-000</td>
			<td></td>
		</tr>
		<tr>
			<td>&#956;PG 101 laser writer</td>
			<td>Heidelberg Instruments</td>
			<td>HI 1128</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic fabrication of core-shell microcapsules carrying human pluripotent stem cell spheroids

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。

通过遵循上述方案，阅读器将能够制造微流体装置并产生携带细胞的微胶囊。 图3A 显示了使用微流体液滴生成制造的最佳和次优微胶囊的示例。PEG-4-Mal的不同配方导致不同形态的胶囊 - 皱纹胶囊与凝胶化不良，机械完整性低且无法承受在搅拌生物反应器中培养有关。在PEG含量为>6%时观察到的光滑胶囊代表了所需的胶囊形态，并且对于细胞培养足够强大。通过将微珠掺入芯中...

Watch this Scientific Journal Video about Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids at JoVE.com

Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids

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。

携带人多能干细胞球体的核壳微胶囊的微流体制造

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and ...

由于这种培养物形成的多能性和分化潜力的提高，该地区被认为是对人类多能干细胞的3D培养物感兴趣的。我们描述了我们的封装策略，该策略导致人类多能干细胞球体的可重复形成。一旦封装，这些干细胞球体就可以在多孔板或搅拌生物反应器中培养。我们的封装策略是优化病虫生存能力和球体形成的高效率。封装的球体可防止与搅拌或摇摆相关的剪切或机械能。干细胞衍生的细胞疗法的开发部分取决于以可扩展，高效和具有成本效益的方式产生所需细胞产品的能力。这里描述的封装技术代表了朝着这个方向迈出的一步。这里描述的封装技术可能适用于从其他细胞到其他多能干细胞的各种细胞类型的3D培养。首先，通过混合4.8克35千道尔顿PEG，10.2毫升密度梯度培养基和19.8毫升DMEM / F-12培养基，制备30毫升两倍核心储备溶液。然后使用0.22微米注射器过滤器过滤此核心溶液。接下来，通过将48.5毫升矿物油和1.5毫升表面活性剂混合，制备50毫升矿物油和3%表面活性剂。然后通过0.22微米注射器过滤器对溶液进行灭菌。接下来，准备1摩尔DTT，1摩尔TEA和1%PF127溶液，如文本手稿中所述。然后将4.5毫升矿物油与3%的表面活性剂和300微升1摩尔DTT混合制备交联剂乳液。将溶液涡旋两分钟后，在20摄氏度的超声波浴中超声处理45至60分钟，并用27号针头将其装入5毫升注射器中。接下来，通过将含有0.5%表面活性剂的矿物油装入具有27号针头的5毫升注射器中来制备屏蔽油溶液。为了制备壳溶液，在400微升DPBS中加入32毫克PEG4马来酰亚胺。然后，加入6微升1摩尔TEA。涡旋溶液2分钟，并通过旋转过滤器离心。将溶液在摇臂上的黑暗中保持30分钟，然后用27号针头装入1毫升注射器中。最后，通过将2000万hPSC重新悬浮在200微升培养基中，并与30微升1%PF127和200微升2X核心溶液混合，制备最终的核心溶液。将一个小搅拌棒插入1毫升注射器中。然后，用27号针头将含有芯溶液的电池加载到注射器中。首先将粘结的PDMS液滴装置，4片20厘米长，1片5厘米长的入口微管，1片10厘米出口管和6片0.5厘米管，放在杀菌光源下30分钟进行灭菌。现在将4个20厘米长的入口微管装入壳中，使用镊子将油，交联剂乳液和解离装置入口安装。然后，将管子的另一端与相应的溶液一起插入注射器针头中。同时，用5厘米的微管连接微流控器件芯的入口和解离器件出口。将 1 根出口管插入流体出口，并将另一端放入收集油箱中。接下来，将设备放在显微镜载物台上并连接管子以避免任何移动。将显微镜对准并聚焦在设备喷嘴上，以进行流量检测。将每个注射器放在不同的注射器泵上并正确固定。根据制造商的协议对文本手稿中提到的流速进行编程，并在所有泵中启动流量。等待每种流体进入设备并填充通道，同时推出剩余的空气。当胶囊形成稳定时，将收集管的另一端放入含有5毫升mTeSR培养基的新锥形管中。收集足够的胶囊后，将其孵育10分钟。位于油相中的胶囊将位于管的顶部，轻轻敲击管子后沉淀到底部。要取回胶囊，首先从收集管中除去油。然后，将胶囊放在100微米孔径的细胞过滤器上，并用大量的培养基清洗过滤器。将过滤器倒置在6孔培养板的孔顶部，并通过使2毫升培养基通过过滤器来收集微胶囊。将干细胞胶囊在37摄氏度的6孔培养皿中孵育5%的二氧化碳。每隔一天更换培养基，方法是将胶囊收集在细胞过滤器过滤器上，用多余的培养基洗涤它们，然后用2毫升培养基将它们重新悬浮到6孔板中的新孔中。通过将4微升的2微摩尔乙锭同源二聚体-1和1微升的4微摩尔钙黄绿素-AM溶液加入2毫升无菌DPBS来进行活力测定。涡流确保完全混合。在细胞过滤器上收集约100个微胶囊，并用无菌DPBS冲洗它们，以使培养基从胶囊中扩散出来。使用1毫升含有乙锭同源二聚体-1和钙黄绿素AM的制备溶液在12孔培养板中再次收集它们，并在37摄氏度下孵育20分钟。在荧光显微镜下观察细胞。使用微流体液滴生成制备的最佳和次优微胶囊显示出胶囊形态的差异，作为壳中PEG4-马来酰亚胺含量的函数。具有明亮边缘的光滑胶囊与所需的机械完整性相关。珠子在胶囊中心的聚集表明核心是水性的，珠子可以自由移动。荧光环空表明存在水凝胶壳，并用于确定壳层厚度。包封H9细胞6小时后的活染色或死染色表明，在包封运行期间，细胞通常达到95%以上的活力。比较包封球体与未包封球体的生长速率和分化潜力。确定包封过程对hPSC没有不利影响。请记住，在核壳和油流之间建立最佳流速比非常重要。如果不这样做，可能会导致胶囊在机械上不坚固，并且在处理过程中可能会破裂。我们想指出的是，这种封装技术适用于人类多能干细胞以外的细胞类型。我们已经使用类似的策略封装了（主要是假设的）癌细胞系和间充质干细胞。目前，我们的团队正在开发下一代微型电容器。这些微捕获具有生物活性，并且可能含有用于包封干细胞分化的交叉因子。

Research

JoVE Journal

Bioengineering

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

科研

生物工程

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

使用粘性图案构建3D纸微流体装置

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

异条件下具有不同刚度的聚乙烯醇-钴-衣康酸水凝胶的人多能干细胞培养

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

管状结构组织工程的核心/壳印刷支架

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

维持静止人类造血干细胞的文化方法

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...