Name: processing of human cardiac tissue toward extracellular matrix self-assembling hydrogel for in vitro and in vivo applications
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Description: Watch this Scientific Journal Video about Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications at JoVE.com

《研究议定书》符合《赫尔辛基宣言》中概述的道德原则。患者提供了知情同意的使用组织的研究目的, 和组织收集的过程是批准的机构审查委员会和伦理委员会 charit-Universitätsmedizin 柏林 (EA4/028/12)。

《研究议定书》符合《赫尔辛基宣言》所述的道德原则, 并得到了 charit 医科大学机构审查委员会和道德问题小组的批准。所有患者都提供了书面的、知情的同意, 以供实验研究使用心脏组织。
1. 人心肌切片的制备
<ol><li>获得左心室心肌 (大小取决于手术类型) 直接从手术室和运输在冷 PBS 在一个无菌容器。</li>
	<li>在无菌条件下, 用无菌手术刀将所有脂肪组织取出10刀片。</li>
	<li>用手术刀切割大约 1 x 1 x 1 cm 的立方体的心肌。</li>
	<li>将立方体置于无菌50毫升管中。 注意: 要暂停, 请执行步骤 1.5, 否则继续步骤2.2。避免重复的冻融循环, 以防止蛋白质退化和组织质量下降。</li>
	<li>在-80 ° c 下储存含有立方体的管子。这些组织可以储存几个月。</li>
</ol>2. 将心肌立方体切片
<ol><li>把装有准备好的立方体的管子从冰箱里拿出来。</li>
	<li>在冰上运送到低温。</li>
	<li>将物体和燃烧室温度设置为-15 ° c。 注意: 正确的温度是保证完美切片的关键。</li>
	<li>冷却空50毫升管和邮票在房间里或在干冰。</li>
	<li>在冰冷的邮票上加入一层 (约 0.5-1.0 毫升) 的 cryosection 介质, 并让它冻结, 直到它是白色和固体。</li>
	<li>加入第二层 cryosection 培养基, 并将心肌立方体放置在这层上。</li>
	<li>请确保 cryosection 培养基和心肌完全冻结, 然后再继续。 注意: 当样品和 cryosection 培养基不结冰时, 切片将会受损。完全冷冻的 cryosection 介质由液体和透明变为固态、白色和非透明。如果立即从步骤1.4 继续, 心肌立方需要至少30分钟至1小时才能完全冻结。当组织被冻结, 它不应该变形, 当触摸/处理与镊子。</li>
	<li>将冰冻心肌的印记插入支架, 拧紧所有螺钉。</li>
	<li>修剪组织的表面直到获得均匀的切片表面。</li>
	<li>将切片厚度设置为300µm。</li>
	<li>冰冻心肌切片自动切片。这保证了一致的切口部分。大约 30-40 部分可以从一个立方体切片。</li>
	<li>在冷50毫升管中松散切片后放置每片。 注意: 请勿按下切片或紧密包装。大约40片可以装在一个管。</li>
	<li>关闭填充管和放置在冰上。 注意: 要暂停使用步骤 2.14, 否则请继续执行步骤3.2。</li>
	<li>在-80 ° c 贮存心肌切片。切片可以存储几个月。</li>
</ol>3. 人心肌切片的细胞
<ol><li>采取必要数量的50毫升管包含心肌部分出-80 ° c 冰箱和运输冰。</li>
	<li>将所有50毫升管子的冰冻切片从步骤3.1 转移到一个无菌烧杯中。大约40片被添加到烧杯每50毫升管。烧杯应为50毫升管体积的三倍;例如,对于一个50毫升管的细胞使用150毫升的烧杯。</li>
	<li>添加50毫升溶解液 (10 毫米三, 0.1% 瓦特/五 EDTA, pH 7.4 在 H2O) 每50毫升的心肌切片管。</li>
	<li>在室温下连续 100-150 rpm, 在无菌烧杯中摇动心肌切片, 持续2小时。</li>
	<li>用粗糙的过滤器将液体与组织切片进行应变。用钝镊子将组织切片转移到新的无菌烧杯中。增加50毫升的 0.5% sds 在 PBS 每初始50毫升心肌切片管 (例如,使用100毫升的 sds 溶液时, 两个50毫升的心肌部分的管正在细胞)。</li>
	<li>在室温下连续摇动6小时, 在 100-150 rpm。</li>
	<li>用粗糙的过滤器将液体与组织切片进行应变。用钝镊子将组织切片转移到一个新的无菌烧杯中, 用50毫升 PBS 清洗3次, 每次10分钟, 同时晃动 150 rpm。接下来, 在 pbs 中用1% 青霉素/链霉素和1% 制 (pbs-P/S-N) 在 100-150 rpm 和4° c 的同时进行夜间清洗。 注: 彻底清洗是至关重要的完全清除任何残留 SDS。当 ECM 与细胞结合使用时, SDS 的残留痕迹是有毒的。</li>
	<li>取出洗涤液, 并添加25毫升预热 FBS (37 ° c) 与1% 青霉素/链霉素和1% 制的细胞切片每初始50毫升管心肌切片。</li>
	<li>在37° c 孵育3小时。在这一步, 任何剩余的 DNA 从母体中移除, 由于 FBS 的内在 dnasei 活动。</li>
	<li>将切片转移到新的无菌烧杯中, 用50毫升 PBS 清洗3次, 每次10分钟, 同时晃动 150 rpm。 注: ECM 切片可在4° c 下贮存数天。但是, 建议立即进行。</li>
</ol>4. 加工细胞 cECM 粉末
<ol><li>将 ECM 切片松散地放在一个新的无菌6井板上。在-80 ° c 和 lyophilize 中冻结 ECM, 在冻中2天。 注意: 每个切片可以放置一个以上。安排切片, 完全覆盖井面。重叠的切片不会影响随后的粉碎成功。</li>
	<li>对于 ECM 切片的粉碎, 请使用特定的铣管 (参见材料表), 其中包含 1.4 mm 陶瓷珠。用无菌钝镊子将管子松散地用冻干的 ECM 填满。为了获得最佳结果, 建议在 10-20 毫克之间进行 ECM 的总重量。 注: 如果 ECM 在铣管中太紧, 粉碎过程将受到负面影响。</li>
	<li>用液氮把管子冻住。 警告:液氮的温度极低, 佩戴适当的个人防护。</li>
	<li>
将冷冻管插入铣床并粉碎 ECM。
	<ol>
<li>设置三十年代和最大速度的持续时间并启动设备。</li>
		<li>完成后, 取出试管, 再在液氮中再次凝固。</li>
		<li>重复五次。</li>
	</ol>
</li>
	<li>通过添加1毫升 ddH2O 每管和震动或漩涡, 为完整的磷清洗微粒从管。通过一个200µm 网格过滤到50毫升管的异构解决方案。</li>
	<li>将管在-80 ° c 或液氮中冷冻。</li>
	<li>用0.22 µm 过滤器更换管的标准盖子。这种过滤器盖子防止粉末流出管, 但允许空气循环为水蒸发。</li>
	<li>插入冻和 lyophilize 中的管子, 将水从步骤中取出 (步骤 4.5) 注: 冻干在这一步可能需要长达3天。</li>
	<li>将粉末贮存在-80 ° c, 直至进一步处理或继续执行步骤5.1。</li>
</ol>5. 基于酶的 cECM 微粒子的均匀化
<ol><li>将猪胃蛋白酶溶于 0.01M HCl (pH 值 2.0), 浓缩1毫克/毫升。在旋转振动筛上放置室温, 直到完全溶解。</li>
	<li>溶解在胃蛋白酶溶液中的冻干人 ECM 粉末 (步骤 4.8), 最终浓度为10毫克/毫升。通过移彻底混合。总容量取决于所需的 ECM 解决方案的数量。例如, 在1毫升胃蛋白酶溶液中溶解10毫克 ECM 粉末。 注: 溶解颗粒可通过温和的涡流 (1000-1500 RPM) 来支持。剩余的微粒会对消化产生负面影响。</li>
	<li>将溶液转移至2毫升管, 每管1毫升。</li>
	<li>把管子放在一个48小时27° c 和 1200 rpm 的管子振动器中。</li>
	<li>把管子从振动筛中取出, 放在冰上或预冷管架上。</li>
	<li>将冷 10x PBS (pH 7.4) 的 ecm 溶液体积的1/9 与1/10 的冷管中的冷0.1M 氢氧化钠的 ecm 溶液体积和冰的位置混合。这种混合物将抵消消化的 ECM 溶液和不可逆转地钝化胃蛋白酶。</li>
	<li>将 ECM 解决方案添加到中和混合物中, 并通过移或涡流进行彻底混合。</li>
	<li>使用 1x PBS (pH 值 7.4) 将 ECM 设置为所需浓度。溶液的 ph 值可以用 ph 值纸检查。 注:10 毫升消化 ECM 解决方案的示例计算: 添加10毫克猪胃蛋白酶到10毫升 HCl (pH 2.0)。 加入100毫克 ECM 粉末到胃蛋白酶溶液。 注: 中和10毫升消化 ECM 溶液的实例计算, 并将浓度设置为8毫克/毫升: 最终= c开始x 卷开始/c最终= 10 毫克/毫升 x 10 毫升/8 毫克/毫升 = 12.5 毫升 10xPBS = 卷开始/9 = 1/9 从10毫升 = 1.11 毫升 10x PBS 氢氧化钠= 卷开始/10 = 1/10 从10毫升 = 1 毫升 卷1xPBS = 卷最终-卷开始-卷10xPBS卷氢氧化钠= 12.5 毫升-10 毫升-11 毫升-1 毫升 = 0.39 毫升</li>
</ol>

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作者没有什么可透露的。

在制备人体心肌 ECM 时, 其目的是实现以下目标: 去除相关的免疫细胞材料, 保护 ecm 的完整性和生物活性, 不育, 最终产品的非毒性, GMP-过程相容性, 以及产品在处理方面的适用性。将我们的3步细胞协议与进一步加工成微粒或自组装水凝胶结合, 获得了具有特定生物活性的人体心脏 ECM 材料, 易于处理, 并具有多个应用程序在体外和在体内, 类似于从几个动物种类中显示的 ECM 产品<sup class=...

细胞外基质 (ECM) 不仅提供结构支持, 而且对生物细胞和组织功能1也很重要。在心脏, ECM 参与调节病理反应, 如纤维化, 炎症, 血管生成, 心肌收缩功能和生存能力, 和居民祖细胞的命运。除了其主要成分-纤维糖蛋白, 多糖, 和蛋白-它包含了一系列分泌生长因子, 细胞因子, 和膜囊泡含有核酸和蛋白质2,3。
最近已经清楚的是, 脱细胞 ECM 制剂不仅对研究细胞基质相互作用, 而且对潜在的治疗细胞基础的应用是非常宝贵的。为治疗性细胞产品或工程组织提供适当环境的重要性现已得到广泛承认。试图结合细胞悬浮液或活性化合物与定义的 biopolymeric 凝胶4,5,6或与小鼠肉瘤细胞分泌的蛋白鸡尾酒 (即,基质, Geltrex)7. 然而, 前者的生物活性有限, 后者在 GMP 级别的过程中存在问题, 且缺乏心脏 ECM (cECM)8、9、10、的组织特异性生物活性.11,12,13。
心肌的细胞先前是通过冠状动脉血管灌注的整个心脏,14,15。虽然这是可能的在动物的心脏, 完整的人的心脏是很少可利用的。因此, 一个浸泡过程, 允许处理在手术室获得的组织样本受到青睐。我们的 "3 步" 协议包含3单独的孵化步骤, 即溶解, 增溶, 和 DNA 去除。它产生人心肌 ECM 与主要保存蛋白质和多糖构成16,17。这些 cECM 切片允许体外研究细胞-基质相互作用, 但不适合潜在的人类规模的治疗应用。然后, 制造过程被扩展到生产冻干 cECM 微粒或 cECM 水凝胶18。
该协议允许从外科标本中获得的人心肌细胞, 保留心肌细胞外基质 (ECM) 的主要成分及其生物活性。在细胞基质相互作用的实验研究中, 或在细胞基心肌再生方法需要合适的环境时, 推荐使用具有组织特异性生物活性的人心脏 ECM。原则上, 也可以将此协议与 GMP 级别的条件相适应, 以便在将来的治疗应用中使用经过处理的 cECM 是可行的。

<table><tbody><tr><td>Balance</td><td>DR Precisa, Dietikon, Switzerland</td><td>Precisa XR 205SM</td><td></td></tr><tr><td>Blades Nr.10 Skalpell Nr.3</td><td>InstrumenteNRW, Erftstadt, Germany</td><td>SK-10-004</td><td></td></tr><tr><td>Cell culture plates (6-well)</td><td>Greiner, Frickenhausen, Germany</td><td>657160</td><td></td></tr><tr><td>Cryostat CM</td><td>Leica, Wetzlar, Germany</td><td>3050S</td><td></td></tr><tr><td>EDTA</td><td>Carl Roth, Karlsruhe, Germany</td><td>8043.3</td><td></td></tr><tr><td>Eppendorf reaction tubes (1.5 or 2 ml)</td><td>Greiner, Frickenhausen, Germany</td><td>616201, 623201</td><td></td></tr><tr><td>Falcon 15ml, 50ml</td><td>Greiner, Frickenhausen, Germany</td><td>188271, 227270</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Biochrome, Berlin, Germany</td><td>S 0115</td><td></td></tr><tr><td>Freeze Dry System</td><td>Labconco, Kansas City, USA</td><td>7670520</td><td></td></tr><tr><td>Freezer (-80&amp;deg;C)</td><td>Thermo Scientific, Waltham, MA, USA</td><td>Forma 900 Series</td><td></td></tr><tr><td>HCl</td><td>Carl Roth, Karlsruhe, Germany</td><td>281.1</td><td></td></tr><tr><td>Microtome Blades Type 819</td><td>Leica, Wetzlar, Germany</td><td>14035838925</td><td></td></tr><tr><td>Minilys Homogeniser</td><td>PEQLAB Biotechnologie GmbH, Erlangen, Germany</td><td>91-PCSM</td><td></td></tr><tr><td>NaOH</td><td>Carl Roth, Karlsruhe, Germany</td><td>K021.1</td><td></td></tr><tr><td>Nystatin</td><td>PAN Biotech, Aidenbach, Germany</td><td>P06-07800</td><td></td></tr><tr><td>PBS</td><td>Thermo Scientific, Waltham, MA, USA</td><td>14190-094</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Life Technologies, Darmstadt, Germany</td><td>15140122</td><td></td></tr><tr><td>Pepsin</td><td>Sigma-Aldrich, Taufkirchen, Germany</td><td>P6887-1G</td><td></td></tr><tr><td>Precellys Keramik-Kit 1.4 mm</td><td>Peqlab Biotechnolgie, Erlangen, Germany</td><td>91-PCS-CK14</td><td></td></tr><tr><td>Rotamax 120 Plate shaker</td><td>Heidolph, Schwabach, Germany</td><td>544-41200-00</td><td></td></tr><tr><td>SDS</td><td>Carl Roth, Karlsruhe, Germany</td><td>CN30.3</td><td></td></tr><tr><td>Stereo microscope</td><td>Leica, Wetzlar, Germany</td><td>M125</td><td></td></tr><tr><td>Steriflip-GP, 0,22 &amp;micro;m</td><td>Merck Millipore, Darmstadt, Germany</td><td>SCGP00525</td><td></td></tr><tr><td>Stuart analogue rocker &amp;amp; roller mixers</td><td>Sigma-Aldrich, Taufkirchen, Germany</td><td>Z675113-1EA</td><td></td></tr><tr><td>Tissue Tek O.C.T compound</td><td>Hartenstein, Wurzburg, Germany</td><td>TTEK</td><td></td></tr><tr><td>Transfusion set 200&amp;micro;m</td><td>Sarstedt, N&amp;uuml;mbrecht, Germany</td><td>798.200.500</td><td></td></tr><tr><td>TRIS</td><td>Carl Roth, Karlsruhe, Germany</td><td>5429.3</td><td></td></tr><tr><td>vedena Skalpellgriff Fig. 3, Standard, 125 mm</td><td>Medical Highlights, Rohrdorf, Germany</td><td>CV102-003</td><td></td></tr><tr><td>Vortex-Genie2</td><td>Scientific Industry, New York, USA</td><td>SI-0256</td><td></td></tr></tbody></table>

processing of human cardiac tissue toward extracellular matrix self-assembling hydrogel for in vitro and in vivo applications

脱细胞细胞外基质制剂对研究细胞基质相互作用和促进再生细胞治疗的应用非常有用。一些商业胞外基质产品可作为水凝胶或膜, 但这些不具有组织特异性的生物活性。由于灌注细胞通常是不可能与人类心脏组织, 我们开发了3步浸泡细胞过程。在手术过程中获得的人心肌切片首先采用无洗涤剂高裂解缓冲液, 然后用离子洗涤剂、十二烷基硫酸钠进行孵化, 并利用其内在 dnasei 活性来完成。胎儿牛血清。这项技术的结果是在心脏细胞外基质的无细胞板, 主要保存纤维组织结构和生物成分, 这表明, 提供了具体的环境线索, 以心电池群体和多能干细胞细胞.心脏胞外基质片可进一步加工成微粒粉, 无需进一步化学修饰, 或者, 通过短期胃蛋白酶消化, 保存成自组装的心脏细胞外基质水凝胶。活性.

3步的人心肌细胞的协议, 导致了近完全去除细胞材料, 同时保留了关键的 ecm 成分和纤维结构的 ecm。细胞后, 通过颜色的变化 (图 1A), 明显去除组织中的细胞。组织学分析与 H & E 和马尾松三污渍显示完全没有剩余完整的细胞 (图 1B)。单个 ECM 成分的定量分析显示, 单独的 DNA 清除和更好地保存总胶原蛋白、弹力蛋白和多糖, 与 SDS (<...

Watch this Scientific Journal Video about Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications at JoVE.com

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

该协议描述了人类心肌的完整细胞, 同时保留了其胞外基质成分。细胞外基质的进一步处理导致微粒和细胞自组装水凝胶的生产。

人心脏组织对细胞外基质自组装水凝胶的处理体外和体内应用

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

processing-human-cardiac-tissue-toward-extracellular-matrix-self

Research

JoVE Journal

Developmental Biology

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

9.0K 次观看.  corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health. 该协议描述了人类心肌的完整细胞, 同时保留了其胞外基质成分。细胞外基质的进一步处理导致微粒和细胞自组装水凝胶的生产。

视频: Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

心肌组织工程生物衍生的注射材料的制备

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

科研

生物工程

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering 

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

心肌在心脏组织工程中的纤维蛋白凝胶的封装

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

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 ...

Custom Engineered Tissue Culture Molds from Laser-etched Masters

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue shape. Manipulating tissue shape on the micrometer to centimeter scale can direct cell alignment, alter effective mechanical properties, and address limitations related to nutrient diffusion. In addition, the vessel in which a tissue is prepared can impart mechanical constraints on the tissue, resulting in stress fields that can further influence both the cell and matrix structure. Shaped tissues with highly reproducible dimensions also have utility for in vitro assays in which sample dimensions are critical, such as whole tissue mechanical analysis.
This manuscript describes an alternative fabrication method utilizing negative master molds prepared from laser etched acrylic: these molds perform well with polydimethylsiloxane (PDMS), permit designs with dimensions on the centimeter scale and feature sizes smaller than 25 &#181;m, and can be rapidly designed and fabricated at a low cost and with minimal expertise. The minimal time and cost requirements allow for laser etched molds to be rapidly iterated upon until an optimal design is determined, and to be easily adapted to suit any assay of interest, including those beyond the field of tissue engineering.

Herein we present a rapid, facile, and low-cost method for fabricating custom polydimethylsiloxane molds that can be used for producing hydrogel-based engineered tissues with complex geometries. We additionally describe results from mechanical and histological assessments conducted on engineered cardiac tissues produced using this technique.

由激光蚀刻大师定制的组织培养模具

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue ...

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

倒置方向下整个人的心脏在加压袋内的去细胞化

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

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 ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

在微流体平台内开发 3D 组织人类心脏组织

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

人诱导多能干细胞来源的心脏球体的生成、高通量筛选和生物样本库

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

3D Human Myocardial Tissue Generation Using Melt Electrospinning Writing of Polycaprolactone Scaffolds and hiPSC-Derived Cardiac Cells

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and regenerative medicine. This protocol describes the stepwise fabrication of 3D myocardial tissues with advanced mimicry of native cardiac structure by combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds with fibrin hydrogels and human induced pluripotent stem cell (hiPSC)-derived cardiac cells. The process involves embedding a mixture of cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (hiPSC-CFs) within a fibrin matrix to create mini-tissues, with structural support provided by MEW-generated scaffolds. These fibrillar scaffolds are fabricated at the micro- to nanoscale, allowing for precise control over fiber architecture, which plays a key role in organizing cell distribution and alignment. Meanwhile, the fibrin matrix promotes cell viability and mimics the extracellular environment. Characterization of the generated tissues reveals well-organized sarcomeres within hiPSC-CMs, along with stable contractile activity. The tissues demonstrate consistent spontaneous beating as early as two days post-seeding, with sustained functionality over time. The combination of hiPSC-CFs with hiPSC-CMs enhances the structural integrity of the tissues while supporting long-term cell viability. This approach offers a reproducible, adaptable, and scalable method for creating biomimetic cardiac tissue models, providing a versatile platform for preclinical drug testing, mechanistic studies of cardiac disease, and potential regenerative therapies.

A reproducible method is presented to generate 3D myocardial tissues combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds and fibrin hydrogels with hiPSC-derived cardiomyocytes and fibroblasts. This technique offers precise control over scaffold architecture and can be applied in preclinical drug testing and cardiac disease modeling.

使用聚己内酯支架和 hiPSC 衍生的心肌细胞的熔融静电纺丝写入生成 3D 人体心肌组织

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

Murine 模型心肌梗塞后 MSC 加载注射水凝胶的心内移植

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...