Name: 3d printing of in vitro hydrogel microcarriers by alternating viscous-inertial force jetting
Uploaded: 2021-04-21T13:00:00
Description: Watch this Scientific Journal Video about 3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting at JoVE.com

本研究由北京市自然科学基金（3212007）、清华大学计划科研计划（20197050024）、清华大学春风基金（20201080760）、国家自然科学基金（51805294）、国家重点研发计划（2018YFA0703004）和111工程（B17026）共同资助。

1. 细胞培养
<ol><li>用10%胎牛血清（FBS），1%非必需氨基酸溶液（NEAA），1%青霉素G和链霉素以及1%谷氨酰胺补充剂作为A549细胞的培养基补充高葡萄糖Dulbecco的改良最低必需培养基。</li>
	<li>在37°C和5%CO 2的CO2培养箱中培养A549细胞
</li>
	<li>解离细胞，使用胰蛋白酶以约80%汇合度进行传代培养。<ol>
<li>使用3mL胰蛋白酶在37°C下处理T75培养瓶中的细胞3分钟，然后加入6mL培养基以停止胰蛋白...

<ol>
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这里描述的方案为制备多种类型的水凝胶微载体和随后的细胞接种提供了说明。与微流控芯片和喷墨打印方法相比，AVIFJ构建微载体的方法具有更大的灵活性和生物相容性。独立的喷嘴使各种轻质喷嘴（包括玻璃微量移液器）可用于这些印刷系统。高度可控的加工使储液器体积、内径和打印头形状等参数可以自由调整。此外，一次性喷嘴便于在多种材料之间切换进行灭菌，从而避免了重复使用的?...

微载体是直径为60-250μm，比表面积较大的微球，通常用于细胞的大规模培养1，2。它们的外表面为细胞提供了丰富的生长位点，内部为空间增殖提供了支撑结构。球形结构还为监测和控制参数提供了便利，包括pH，O2以及营养物质和代谢物的浓度。当与搅拌罐生物反应器结合使用时，与传统培养物相比，微载体可以在相对较小的体积内实现更高的细胞密度，从而为实现大规模培养提供了一种经济有效的方法3。微载体培养技术已成为细胞学研究的主要技术之一，在干细胞、肝细胞、软骨细胞、成纤维细胞和其他结构的大规模扩增领域取得了很大进展4。它们也被发现是理想的药物输送载体和自下而上的单位，因此在临床药物筛选和 体外 组织工程修复中发挥着越来越重要的作用5。
为了满足不同场景下的力学性能要求，开发了多种类型的水凝胶材料，用于微载体的建造6，7，8，9，10，11。海藻酸盐和透明质酸（HA）水凝胶是两种最常用的微载体材料，因为它们具有良好的生物相容性和交联性12，13。海藻酸盐可以很容易地被氯化钙交联，其机械性能可以通过改变交联时间来调节。酪胺共轭HA通过过氧化氢和辣根过氧化物酶14催化的酪胺部分的氧化偶联而交联。胶原蛋白由于其独特的螺旋结构和交联纤维网络，经常被用作佐剂混合到微载体中，以进一步促进细胞附着15，16。
目前制备微载体的方法包括微流控芯片，喷墨打印和电喷雾17，18，19，20，21，22，23。微流控芯片已被证明在生产大小均匀的微载体方面具有快速高效的特性24。然而，该技术依赖于复杂的流道设计和制造工艺25。喷墨打印过程中的高温或过大的挤出力，以及电喷雾方法中的强电场，可能会对材料的性能产生不利影响，尤其是其生物活性19。此外，当应用于各种生物材料和直径时，这些方法中使用的定制喷嘴导致加工复杂性有限，成本高，灵活性低。
为了提供一种方便的微载体制备方法，应用了一种称为交替粘性惯性力喷射（AVIFJ）的3D打印技术来构建水凝胶微载体。该技术利用垂直振动过程中产生的向下驱动力和静压来克服喷嘴尖端的表面张力，从而形成液滴。在打印过程中，小的快速位移不是强烈的力和热条件，而是直接作用于喷嘴，对生物墨水的物理化学性质产生轻微影响，并对生物活性材料表现出极大的吸引力。利用AVIFJ方法，成功形成了直径为100-300 μm的多种生物材料的微载体。此外，微载体进一步证明可以很好地结合细胞，并为粘附的细胞提供合适的生长环境。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A549 cells</td>
			<td>ATCC</td>
			<td>CCL-185</td>
			<td>Human non-small cell lung cancer cell line</td>
		</tr>
		<tr>
			<td>Bright field microscope</td>
			<td>Olympus</td>
			<td>DP70</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>TI-FL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum, FBS</td>
			<td>BI</td>
			<td>04-001-1ACS</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>SIGMA</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass micropipettes</td>
			<td>sutter instrument</td>
			<td>b150-110-10</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>GIBCO</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>H-DMEM</td>
			<td>GIBCO</td>
			<td>11960-044</td>
			<td>Dulbecco&#39;s modified eagle medium</td>
		</tr>
		<tr>
			<td>Horseradish peroxidase powder</td>
			<td>SIGMA</td>
			<td>P6782</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic agent</td>
			<td>3M</td>
			<td>PN7026</td>
			<td>Follow the manufacturer&#39;s instructions and use after dilution</td>
		</tr>
		<tr>
			<td>Micro-forge device</td>
			<td>narishige</td>
			<td>MF-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids, NEAA</td>
			<td>GIBCO</td>
			<td>11140-050</td>
			<td>non-essential amino acids</td>
		</tr>
		<tr>
			<td>Penicillin G and streptomycin</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>SIGMA</td>
			<td>P5731-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Puller</td>
			<td>sutter instrument</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium alginate</td>
			<td>SIGMA</td>
			<td>A0682</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>GIBCO</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Type I collagen solution from rat tail</td>
			<td>SIGMA</td>
			<td>C3867</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d printing of in vitro hydrogel microcarriers by alternating viscous-inertial force jetting

微载体是直径为60-250μm且比表面积较大的微球，通常用作大规模细胞培养的载体。微载体培养技术已成为细胞学研究的主要技术之一，在大规模细胞扩增领域得到普遍应用。微载体也被证明 在体外 组织工程构建和临床药物筛选中发挥着越来越重要的作用。目前制备微载体的方法包括微流控芯片和喷墨打印，它们通常依赖于复杂的流道设计，不兼容的两相界面和固定的喷嘴形状。这些方法面临着复杂的喷嘴加工，不方便的喷嘴更换以及应用于多个生物墨水时挤出力过大的挑战。在这项研究中，应用了一种称为交替粘性 - 惯性力喷射的3D打印技术，以构建直径为100-300μm的水凝胶微载体。随后将细胞接种在微载体上以形成组织工程模块。与现有方法相比，该方法为各种生物活性材料提供了自由的喷嘴尖端直径，灵活的喷嘴切换，自由控制打印参数和温和的打印条件。

制造了不同收敛速率和直径的打印头，以实现多种类型的材料的打印。随着拉力的增加而获得的喷嘴如图 1B所示。喷嘴分为三个区域：储液槽（III），收缩（II）和打印头（I）。储液罐是喷嘴的未加工部分，其中液体提供静压和生物墨水输入以进行打印。收缩区域是产生向下驱动力的主要部分。拉力对打印头有显著影响，显示出较低的收敛率和扩展的拉力。较窄和较长的打?...

Watch this Scientific Journal Video about 3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting at JoVE.com

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

这里介绍的是一种温和的3D打印技术，由交替的粘性惯性力驱动，以实现水凝胶微载体的构建。自制喷嘴具有灵活性，可轻松更换不同的材料和直径。可以获得直径为50-500μm的细胞结合微载体并收集以进行进一步培养。

通过交替粘性 - 惯性力喷射进行 体外 水凝胶微载体的3D打印

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...

我们提供温和的生物打印工艺，用于制备具有良好可控性和生物相容性的微载体。在被细胞包封后，载体还可用于体外细胞扩增和功能微组织构建。在印刷过程中，有限的位移，而不是在类似条件下作用于喷嘴的严重力，最大限度地保持生物墨水的原始物理/化学特性。除了传统的微载体外，还可以构建具有内部细胞分布或三聚糖苷包封胶囊结构的单元并将其应用于不同组织结构的组装。对于喷嘴准备，根据制造商的说明将玻璃微量移液器加载到拉拔器上，并为拉拔器设置拉拔参数。使用微孔切断指定直径的喷嘴，以获得实验的特定尖端直径，并将喷嘴在酒精中灭菌五分钟。然后，用无菌水冲洗喷嘴三次，以除去任何残留的酒精。为了制备水凝胶生物墨水，将灭菌的4%海藻酸钠储备溶液稀释在0.9%氯化钠中以0.5%，1，1.5和2%的重量体积浓度。对于微滴形成，将五毫升生物墨水装入一次性无菌注射器中，并将注射器安装到注射器泵上。使用一毫米内径软管，连接注射器和打印喷嘴。拧紧夹紧螺钉以将喷嘴固定到位，并快速按下注射器泵以将生物墨水加载到喷嘴中。设置信号发生器参数，并预设振动的运动路径和触发模式，以按需下降。然后按照预先设计的图案打印液滴。对于微载体的形成，向培养皿中加入五毫升交联溶液，并将培养皿置于印刷喷嘴作为基材下方。打印后，交联微载体三分钟，然后将微载体悬浮液转移到离心管中。然后，通过离心富集微载体，并将其重悬于适当的培养基中，每毫升约600个微载体。在接种之前，用5毫升补充有10微摩尔细胞追踪器绿色CMFDA染料的无血清培养基替换A549细胞培养物的上清液，在细胞培养箱中孵育30分钟。在孵育结束时，用新鲜培养基替换染料溶液，并以每毫升培养基1.6倍10的密度重悬细胞至6个细胞。向细胞中加入一毫升微载体悬浮液，向低贴壁六孔培养板的每个孔中加入一毫升A549细胞。将板以每分钟30转的速度放在细胞培养箱中的振荡器上。在孵育结束时，从振荡器中取出板，让微载体沉降30分钟，然后通过明场和共聚焦荧光显微镜观察和测量打印喷嘴，培养皿和微载体接种的细胞。使用30微米尖端喷嘴，可以将多种类型的生物墨水（包括PBS，1.5%海藻酸盐和1.5%明胶）稳定地打印到培养皿上。随着油墨粘度的增加，印刷过程变得越来越困难，需要更大的驱动力来获得更大的液滴。打印参数可以设置为以特定的2D排列打印液滴，如本打印实验中所观察到的那样。喷嘴尖端的直径直接影响可打印的微载体的尺寸。正如在这些明场和共聚焦图像中观察到的那样，A549细胞在培养两天后粘附在藻酸盐 - 胶原微载体上。六天后，A549电池几乎完全覆盖了微载体表面。按照该过程，我们用壳聚糖涂覆微载体表面，并使用葡萄糖酸盐溶解海藻酸盐凝胶以形成囊性结构，该囊性结构可用于构建肺泡等空心结构。制备微载体的印刷工艺相当温和，因此，该方法可广泛应用于含细胞微载体的印刷。

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3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

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