Name: pancreatic tissue-derived extracellular matrix bioink for printing 3d cell-laden pancreatic tissue constructs
Uploaded: 2019-12-13T15:00:00
Description: Watch this Scientific Journal Video about Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs at JoVE.com

这项研究得到了韩国政府资助的国立研究基金会（NRF）的生物与医疗技术开发项目（2017M3A9C6032067）和"ICT康力创意计划"（IITP-2019-2011-1-00783）的支持。由IITP（信息与通信技术规划与评估研究所）监督。

猪胰腺组织是从当地的屠宰场收集的。动物实验得到了韩国首尔亚山医学中心机构动物护理和使用委员会（IACUC）的批准。
1. 组织去细胞化
<ol><li>准备脱细胞化的解决方案。 注：所有溶液制剂中使用的1倍磷酸盐缓冲盐水（PBS）通过向10x PBS中加入蒸馏水稀释。<ol>
<li>对于 1% Triton-X 100 溶液，使用磁搅拌棒在 150 rpm 下搅拌 6 小时，在 900 mL 的 1x PBS 中溶解 100 mL 的 100% Triton-X 10...

<ol>
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该协议描述了pdECM生物油墨的开发以及使用3D细胞打印技术构建3D胰腺组织结构。要概括3D工程组织构造中目标组织的微环境，生物油墨的选择至关重要。在以前的研究中，我们验证了组织特异性的dECM生物油墨有利于促进干细胞分化和增殖10。与合成聚合物相比，dECM可以作为一个有利于细胞的环境，因为组织特异性的组成和结构11。因此，在dECM中主要部件的高保?...

最近，胰腺胰岛移植被认为是对1型糖尿病患者的一种有前途的治疗方法。该手术的相对安全性和最小侵入性是这种治疗1的很大优势。然而，它有一些限制，如分离胰岛的低成功率和免疫抑制药物的副作用。此外，由于恶劣的环境2，移植后移植胰岛的数量稳步减少。各种生物相容性材料，如藻酸盐、胶原蛋白、聚（乳胶共甘油酸）或聚乙烯乙二醇（PEG）已应用于胰岛移植，以克服这些困难。
3D细胞打印技术以其巨大的潜力和高性能在组织工程中不断涌现。不用说，生物油墨被称为提供合适的微环境，并促成印刷组织结构中细胞过程改善的重要成分。大量的剪切变薄水凝胶，如纤维蛋白、藻酸盐和胶原蛋白被广泛用作生物油墨。然而，与原生组织3中的细胞外基质（ECM）相比，这些材料表现出缺乏结构、化学、生物和机械的复杂性。微环境提示，如胰岛和ECM之间的相互作用，是增强胰岛功能的重要信号。脱细胞化ECM（dECM）可以重新创建各种ECM成分的组织特异性组合物，包括胶原蛋白、糖氨基甘油（GAG）和糖蛋白。例如，保留其周围E意象带的初级胰岛（例如，I型、III型、IV型、V型和VI型胶原蛋白、拉米宁和纤维素）表现出低细胞凋亡和更好的胰岛素敏感性，这表明组织特异性细胞-基质相互作用对于增强其与原始组织4类似的功能能力非常重要。
在本文中，我们阐明了制备胰腺组织衍生的去细胞外基质（pdECM）生物油墨的方案，为增强胰腺胰岛的活性和功能提供有益的微环境提示，然后使用基于微糊精的生物印刷技术生成3D胰腺组织结构的过程（图1）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Biological Safety Cabinets</td>
			<td>CRYSTE</td>
			<td>PURICUBE 1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Deep Freezer</td>
			<td>Thermo Scientific Forma</td>
			<td>957</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital orbital shaker</td>
			<td>DAIHAN Scientific</td>
			<td>DH.WSO04010</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry oven</td>
			<td>DAIHAN Scientific</td>
			<td>WON-155</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze dryer</td>
			<td>LABCONCO</td>
			<td>7670540</td>
			<td></td>
		</tr>
		<tr>
			<td>Fridge</td>
			<td>SANSUNG</td>
			<td>CRFD-1141</td>
			<td></td>
		</tr>
		<tr>
			<td>Grater</td>
			<td>ABM</td>
			<td>1415605793</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscopes</td>
			<td>Leica</td>
			<td>DMi1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>CRYSTE</td>
			<td>PURISPIN 17R</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Thermo Fisher Scientific</td>
			<td>Multiskan GO</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini centrifuge</td>
			<td>DAIHAN Scientific</td>
			<td>CF-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-Hotplate Stirrers</td>
			<td>DAIHAN Scientific</td>
			<td>SMHS-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-LITE-PR</td>
			<td></td>
		</tr>
		<tr>
			<td>pH benchtop meter</td>
			<td>Thermo Fisher Scientific</td>
			<td>STARA2110</td>
			<td></td>
		</tr>
		<tr>
			<td>Rheometer</td>
			<td>TA Instrument</td>
			<td>Discovery HR-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>DAIHAN Scientific</td>
			<td>VM-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Cirurgical Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissors</td>
			<td>Hirose</td>
			<td>HC.13-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>Korea Ace Scientific</td>
			<td>HC.203-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 mL microcentrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml glass vial</td>
			<td>Scilab</td>
			<td>SL.VI1243</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m cell strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>5 L beaker</td>
			<td>Dong Sung Science</td>
			<td>SDS 2400</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL cornical tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL beaker</td>
			<td>Korea Ace Scientific</td>
			<td>KA.23-08</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL bottle-top vacuum filter</td>
			<td>Corning</td>
			<td>431118</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL plastic container</td>
			<td>LOCK&#38;LOCK</td>
			<td>INL301</td>
			<td></td>
		</tr>
		<tr>
			<td>96well plate</td>
			<td>Falcon</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>DAEKYO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Kimtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic bar</td>
			<td>Korea Ace Scientific</td>
			<td>BA.37110-0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Mortar and pestle</td>
			<td>DAIHAN Scientific</td>
			<td>SC.MG100</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel pipettor</td>
			<td>Eppendorf</td>
			<td>4982000314</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>SPL</td>
			<td>10100</td>
			<td></td>
		</tr>
		<tr>
			<td>pH indicator strips</td>
			<td>Sigma-Aldrich</td>
			<td>1095350001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sieve filter mesh</td>
			<td>DAIHAN Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Decellularization</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x pbs</td>
			<td>Hyclone</td>
			<td>SH30258.01</td>
			<td></td>
		</tr>
		<tr>
			<td>4.7% Peracetic acid</td>
			<td>Omegafarm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>SAMCHUN CHEMICALS</td>
			<td>E0220 SAM</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IPA</td>
			<td>SAMCHUN CHEMICALS</td>
			<td>samchun I0348</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Biosesang</td>
			<td>T1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Biochemical assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1,9-Dimethyl-Methylene Blue zinc chloride double salt</td>
			<td>Sigma-Aldrich</td>
			<td>341088</td>
			<td></td>
		</tr>
		<tr>
			<td>10 N NaOH</td>
			<td>Biosesang</td>
			<td>S2018</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramine T</td>
			<td>Sigma-Aldrich</td>
			<td>857319</td>
			<td></td>
		</tr>
		<tr>
			<td>Chondroitin sulfate A</td>
			<td>Sigma-Aldrich</td>
			<td>C4384</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Supelco</td>
			<td>46933</td>
			<td></td>
		</tr>
		<tr>
			<td>Cysteine-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>C1276</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Merok</td>
			<td>100063</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>410225</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2-EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>SAMCHUN CHEMICALS</td>
			<td>S2097</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Sigma-Aldrich</td>
			<td>p4762</td>
			<td></td>
		</tr>
		<tr>
			<td>P-DAB</td>
			<td>Sigma-Aldrich</td>
			<td>D2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Perchloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>311421</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S5636</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Supelco</td>
			<td>SX0607N</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate(monobasic)</td>
			<td>Sigma-Aldrich</td>
			<td>RDD007</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Sigma-Aldrich</td>
			<td>244511</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioink</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Charicterized FBS</td>
			<td>Hyclone</td>
			<td>SH30084.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Sigma-Aldrich</td>
			<td>P7215</td>
			<td></td>
		</tr>
		<tr>
			<td>Rose bengal</td>
			<td>Sigma-Aldrich</td>
			<td>198250</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
	</tbody>
</table>

pancreatic tissue-derived extracellular matrix bioink for printing 3d cell-laden pancreatic tissue constructs

胰腺胰岛移植是1型糖尿病患者有低血糖和继发性并发症的一种有前途的治疗方法。然而，胰岛移植仍然有几个限制，如移植胰岛由于贫困胰岛移植和恶劣环境而存活率低。此外，从人类多能干细胞中分化的胰岛素生成细胞具有有限的分泌足够的激素的能力，这些激素可以调节血糖水平;因此，迫切需要通过培养具有适当微环境线索的细胞来改善成熟。在本文中，我们阐明了制备胰腺组织衍生的细胞外基质 （pdECM） 生物墨水的协议，以提供一个可增加胰腺胰岛葡萄糖敏感性的有益微环境，然后描述使用基于微糊精的生物印版技术生成3D胰腺组织构建的过程。

胰腺组织的去细胞化 我们开发了制备 pdECM 生物墨水的工艺，以提供胰腺组织特定的微环境，以增强 3D 生物打印组织结构中的胰岛功能（图 2A）。脱细胞化过程后，97.3%的dsDNA被去除，胶原蛋白和GAG等代表性ECM成分与原生胰腺组织相比分别保持在1278.1%和96.9%（图2B）。
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Watch this Scientific Journal Video about Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs at JoVE.com

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

去细胞化细胞外基质（dECM）可以提供合适的微环境线索，以在工程结构中重述目标组织的固有功能。本文阐述了胰腺组织去细胞化、胰腺组织衍生dECM生物油墨评价以及利用生物印刷技术生成3D胰腺组织构造的方案。

用于打印 3D 细胞-拉丹胰腺组织构造的胰腺组织衍生细胞外基质生物墨水

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

pdECM 生物墨水可以使用组织特异性成分和结构为胰岛提供有益的微环境，具有最佳的神经特性，可减少细胞死亡并增加其可打印性。该技术的主要优点是，组织特异性组成可以保存在pdECM生物墨水中，促进3D胰腺组织构造和封装胰岛之间的建设性相声。pdECM生物墨水中胰岛封装的发展可应用于可移植胰腺组织结构的制造，用于治疗1型糖尿病。这种生物墨水可用于扩大可移植结构的应用，以及体外组织模型对糖尿病、糖尿病相关并发症和胰腺癌的应用。对于冷冻猪胰腺组织样品的去细胞化，使用碎液将冷冻胰腺切成一毫米厚的碎片，然后将50克切片组织转移到500毫升塑料容器中。在数字轨道摇床上，以每分钟150次旋转的速度用300毫升蒸馏水清洗组织，约12小时。当浑浊的水消失时，用PBS中1%Triton-X 100的400毫升水代替水84小时。在洗涤剂处理结束时，用400毫升异丙醇孵育组织两个小时，从胰腺中去除剩余的脂肪，然后用400毫升PBS进行24小时洗涤。在洗涤结束时，用4%乙醇中的400毫升0.1%过乙酸消毒脱细胞组织两个小时，然后用400毫升新鲜PBS清洗样品6小时，以去除任何残留的洗涤剂。在洗涤结束时，用钳子将组织件转移到50毫升锥形管中，并在零下80摄氏度下重新冷冻样品一小时。然后用用橡皮筋固定的无绒擦拭，将去细胞化组织冻干四天，覆盖锥形管。为了制备生物墨，将200毫克冷冻干燥pdECM转移到一个新的50毫升锥形管中，并在组织样品中加入20毫克的辣椒和8.4毫升的0.5摩尔醋酸。然后将磁搅拌棒放入管中，以每分钟 300 次旋转的速度进行 96 小时的搅拌孵化。在孵化结束时，使用40微米细胞过滤器和正位移液器将溶液过滤成冰上新管，过滤掉任何未消化的颗粒，并将10X PBS的毫升加入管中。涡旋后，使用氢氧化钠将溶液的pH值调整为7。为了准备具有图案结构的胰腺结构的3D细胞打印的生物墨，用0.4%Trypan Blue染色一个pdECM生物墨水，用玫瑰孟加拉溶液以1比20的比例染色一个等分。然后使用正位移液器将每个生物墨水溶液与中等悬浮小岛溶液以三比一的比例与最终的 1.5% 生物墨水浓度和细胞密度的三倍 10 到三小升等量的细胞混合。对于基于多材料的胰腺组织构造的 3D 细胞打印，将每个生物墨水胰岛混合物加载到配备 25 个仪表喷嘴和 3D 打印的每个生物墨水中，在 18 摄氏度的优化打印条件下，以蓝色和红色交替线的形状进行打印。要将生物墨水交后，将印刷结构在37摄氏度和5%的二氧化碳细胞培养箱中放置30分钟。然后将印刷结构浸入 RPMI 1640 介质中，辅以 10% 胎儿牛血清和每毫升青霉素和链霉素 100 个单位。脱细胞过程后，97.3%的双链DNA被移除，与原生胰腺组织相比，胶原蛋白和糖核糖等代表性细胞基质成分分别保持在1278.1%和96.9%。在此代表性分析中，pdECM 生物墨表现出剪切变薄行为，以 1 秒 1 的剪切速率计算，其值约为 10 帕斯卡/秒，表明生物墨脱显示了通过喷嘴挤出的适当流变特性。此外，当温度达到15摄氏度时，生物墨水的复杂模量开始增加，当温度保持在37摄氏度时，该模量开始迅速增加，表明溶液的溶胶过渡。在生理相关温度下对pdECM生物油墨的动态G素数和G双素数进行了调查，以确保其在打印过程后保持稳定性，从而在频率扫描条件下实现稳定的模量。然后，使用多头打印系统，可以制造各种类型的 3D 结构，例如使用开发的 pdECM 演示 pdECM 的多功能性，用于 3D 生物打印，以组织形式排列协调两种或两种多种类型的活组织。将生物墨水与细胞混合时要温和，以避免细胞死亡和气泡形成，因为气泡的存在会降低打印分辨率并降低细胞的生存能力。3D 胰腺组织结构中封装的胰岛的功能可以通过免疫荧光染色或葡萄糖耐受性测试进行评估。这种pdECM生物墨和三D生物打印技术可用于组织工程领域功能性胰腺组织结构的发展。研究人员在处理锋利的磨衣机和试剂时应佩戴适当的个人防护装备，以防止受伤。

Research

JoVE Journal

Bioengineering

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

科研

生物工程

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

细胞外基质刚性三维肿瘤模型的纵向测量用粒子跟踪Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

生物印刷细胞化构造使用组织特异性水凝胶生物油墨

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

小鼠脂肪组织来源的微血管片段作为血管化单元的分离组织工程

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

肾皮质细胞外基质衍生水凝胶的制备

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Novel Process for 3D Printing Decellularized Matrices

一种新的三维打印解光矩阵处理方法

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...