作者承认以下资金来源:神经科学心血管研究基金和马里兰州干细胞研究基金（2016-MSCRFI-2735）。

1.心肌细胞的制备<ol><li>如上所述，在用基底膜基质包被的6孔板上产生和培养人诱导的多能干细胞（hiPSC）。 </li><li>使用先前描述的方法11,12将hiPSC分化为hiPSC来源的心肌细胞（hiPSC-CM）。 </li><li>分化后第19天，使用2mL胰蛋白酶/ EDTA的胰蛋白酶/ EDTA 0.05％的室温分离5分钟。 </li><li>在光学显微镜下监测心肌细胞以观察细胞解离。 </li><li>使用2mL胰蛋白酶抑制剂中和胰蛋白酶0.0125％。 </li><li>放置分离的心肌细胞，并使用电动吸管填充物将其转移到一个50 mL锥形管中。 </li><li>在室温下以250×g离心细胞悬液5分钟，得到沉淀物。 </li><li>将沉淀物重新悬浮在5mL Roswell Park中补充有B-27（RPMI / B-27细胞培养基）的纪念研究所（RPMI）细胞培养基。 </li><li>移取细胞培养液中的20μL细胞，并用等量的0.4％台盼蓝溶液进行染色。 </li><li>使用自动细胞计数器或手动血细胞计数器来计数和获得细胞悬浮液的浓度和细胞活力。 </li></ol> 2.成纤维细胞的制备<ol><li>启动人心脏成纤维细胞（HCF）（成人心室型）细胞系13 。 </li><li>通过并根据HCF培养方案分离它们13 。 </li><li>移取细胞培养液中的20μL细胞，并用等量的台盼蓝溶液0.4％染色。 </li><li>使用自动细胞计数器或手动血细胞计数器来计数和获得新细胞悬浮液的浓度和细胞活力。 </li></ol> 3.内皮细胞的制备<ol><li>启动人类的伞如静脉内皮细胞（HUVEC）线所述。按照HUVEC培养方案进行传播并分离，如图14所示 。 </li><li>移取细胞培养液中的20μL细胞，并用等量的0.4％台盼蓝溶液进行染色。 </li><li>使用自动细胞计数器或手动血细胞计数器来计数和获得细胞悬浮液的浓度和细胞活力。 </li></ol>共同文化: <ol><li>将三种细胞类型（hiPSC-CM，HCF和HUVEC）混合在RPMI / B-27细胞培养基中，以一个hiPSC-CM:HCF:HUVEC细胞比率在50mL锥形管中产生混合细胞溶液原料70:15:15，浓度为165,000个细胞/ mL。 </li><li>使用多通道移液管将混合细胞溶液分配到每孔200μL或每孔33,000个细胞的超低附着96孔U底板中。 </li><li>将96孔板孵育3天（37℃，5％二氧化碳）ide，95％湿度）。 </li><li>在光学显微镜下检查单个孔的中部和底部混合细胞多细胞球体的存在。 注意:在二维微观投影中，这些球体将呈圆形（ 图1 ）。在细胞接种到超低附着的96孔板后24小时内，球形体应形成。细胞播种到96孔板后48小时内，球体应开始跳动。不跳动的球体不应用于3D生物印刷，以确保最终的3D生物印迹心脏贴片是功能性的。球蛋白对连接蛋白43（Cx43）和肌钙蛋白的免疫荧光可用于评估球状细胞组成和质量15 。 </li></ol> 5.脚手架心脏组织的3D生物印迹<ol><li>将含有球体的板载入无支架的基于球体的3D生物打印机的杂志。 </li><li> 打开3D生物打印机并执行3D生物打印软件。 <ol><li>单击"辅助"，"操作准备"，"检查模式"，然后单击"归位"。 </li><li>单击"开始"下的绿色图标开始球体检查。 </li><li>观察球体直径出现在屏幕上的"（2）Dia（μm）"列下。 </li></ol></li><li>要将平均球体直径估算为最接近的100μm，请单击"初始设置"，并将平均球体直径输入到堆叠Z间距（μm）。 </li><li>单击"总计"，"层"下的数字应自动调整。点击"应用"确认设置。 </li><li>点击"针数组图"，进入"图层编号"，选择所需的3D生物印刷图层，并通过选择单个球体位置，在屏幕左侧的地图上绘制所需的3D生物印刷设计。社区法网k"应用"以确认3D生物印刷设计。 </li><li>点击"针阵检查"开始针阵检查。如果针阵列失焦，则输入值为"OffsetZ（μm）"，范围为0〜500μm，调整摄像机对焦，起始时间为500μm，减少100μm至0μm。 </li><li>单击"检查参数"，"类型1"，并将"直径（μm）"设置为所需的直径范围。将"圆度（％）"和"平滑度（％）"设置为所需的百分比。点击"应用"确认设置。 注意:这里的球体直径为450μm至550μm。 "圆度（％）"和"平滑度（％）"是计算机计算每个球体的评估。这里，用于"圆度（％）"的设置为60％，"平滑度（％）"为70％。 </li><li>单击"存在"并将"直径（μm）"更改为相同的设置在上一步。点击"应用"确认设置。 </li><li>单击"堆叠模式"，然后点击"开始"下的绿色图标，使用3D生物打印机的喷嘴通过真空吸力开始球体拾取，并将球体放置在针阵列中的特定位置。 注意:对于由81个球体（9个球体×9个球体）组成的心脏贴片，3D生物印迹需要约20至30分钟。 </li><li>生物印刷后，使用无菌镊子，拿起包含3D生物印迹贴片的针阵列，并将其转移到含有150 mL RPMI / B-27细胞培养基的250 mL无菌烧杯中。 </li><li>使用针阵列孵育3D生物印迹贴片3天（37℃，5％二氧化碳，95％湿度）。 </li></ol> 6.从针阵列和贴片成熟中去除3D生物印迹贴片<ol><li>孵育3天后，用一只手在针阵列的底部，另一只手在塑料盖上他补丁，轻轻地将塑料盖向上滑动以从针阵列中移除3D生物印迹补丁。 注意:根据需要，可以在本步骤之前将磷酸盐缓冲盐水（PBS）润滑施用于针头，以便更平滑和更容易地去除。 </li><li>使用一对无菌镊子，将3D生物印迹贴片拾起塑料盖，并将贴片和塑料盖转移到含有RPMI / B-27细胞培养基的35 mm培养皿中。 </li><li>使用RPMI / B-27细胞介质中的贴片轻轻摇动塑料盖，镊子仍贴在塑料盖上，将贴片释放到介质中。 </li><li>在光学显微镜下检查3D生物印迹贴片可视化是否打败以确保3D生物印迹贴片功能正常。 </li><li>将三维生物印迹贴片在35毫米的培养皿中孵育3天或更长时间（37℃，5％二氧化碳，95％湿度）。 注意:3天后，球体会熔合在一起，并将孔插入那些针头的补丁不应该再显眼了。 </li></ol>

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作者没有什么可以披露的。

It is important to use beating, functional spheroids for 3D bioprinting. If spheroids are not beating, continuing to use them will invariably result in a non-functional 3D bioprinted patch.
One benefit of this approach is the ability to manipulate the cell content of the patch by varying the total number of cells and the percentage of cardiomyocytes, endothelial cells, and fibroblasts in the spheroids. This allows for many different types of cardiac patches to be printed, with varying histolog...

3D生物印迹有很多不同的方法， 1，2，3 。 3D生物打印通常通过印刷技术1进行分类，例如喷墨生物印刷，微压缩生物印刷，激光辅助生物印刷，方法的组合或更新的方法。 3D生物印迹也可以分为无支架或支架依赖的方法4 。 3D生物印迹的大多数方法是支架依赖的，其中需要生物材料， 例如生物蛋白5或支架6 。然而，支架依赖的3D生物印迹面临许多问题和局限性，如脚手架材料的免疫原性，专有生物蛋白的成本，降解产物的速度和毒性等。 SCAF已经尝试使用球体的无折叠心脏组织工程8 ，有可能克服支架依赖组织工程的这些缺点。然而，正如作者在该论文中所承认的那样，在生物制造过程中难以强力地处理和定位固定位置的球体。伴随使用3D生物印迹和基于球形的组织工程有可能克服这些困难。在本协议中，我们描述了没有其他生物材料的心脏组织的3D生物印迹，仅使用球形形式的细胞。 基于无支架的基于球体的3D生物打印机9具有使用真空抽吸拾取单个球体的能力，并将其定位在针阵列上。 3D生物印刷中针阵列上的定位球体的概念是从古代日本使用针阵（被称为" kenzan "）的灵感来的nese艺术的花卉安排， ikebana。该系统允许球体精确地定位在任何配置中，并导致单个球体在短时间内熔合在一起以产生3D生物印刷组织。因此，该方法允许容易地操纵球体，对于无支架器官生物制造的未来具有潜在的影响。

<table border="0" cellpadding="0" cellspacing="0" width="587">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Geltrex</td>
			<td style="width:157px;">Invitrogen&#160;</td>
			<td style="width:88px;">A1413202</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Trypsin/EDTA 0.05%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">15400054</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Defined Trypsin inhibitor 0.0125%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">R007100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:176px;">RPMI Cell Media</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:88px;">11875-093</td>
			<td style="width:167px;">RPMI supplemented with B27 constitutes HIPSC-CM culture media</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:176px;">B-27 Supplement</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">17504044</td>
			<td style="width:167px;">RPMI supplemented with B27 constitutes HIPSC-CM culture media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Countess Automated Cell Counter</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:88px;">C10227</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:176px;">Human cardiac fibroblasts (adult ventricular type)</td>
			<td style="width:157px;">Sciencell</td>
			<td style="width:88px;">6310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Human umbilical vein endothelial cells</td>
			<td style="width:157px;">Lonza</td>
			<td style="width:88px;">CC-2935</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:176px;">PrimeSurface ultra-low attachment 96-well U-bottom plates&#160;</td>
			<td style="width:157px;">Akita Sumitomo Bakelite Co.</td>
			<td style="width:88px;">MS-9096UZ</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Regenova Bio 3D Printer</td>
			<td style="width:157px;">Cyfuse Biomedical K.K.</td>
			<td style="width:88px;">N/A</td>
			<td>www.cyfusebio.com/en/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Troponin T Antibody</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">701620</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Connexin 43 (Cx43) Antibody</td>
			<td style="width:157px;">Chemicon</td>
			<td>MAB3068</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">ProLong Gold Antifade Mountant with DAPI</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">P36935</td>
			<td></td>
		</tr>
	</tbody>
</table>

creation of cardiac tissue exhibiting mechanical integration of spheroids using 3d bioprinting

该协议描述了仅使用细胞而不使用生物材料的心脏组织的3D生物印迹。首先分离心肌细胞，内皮细胞和成纤维细胞，计数并以所需细胞比例混合。它们在超低附件96孔板中的各个孔中共培养。在3天内，殴打球体。然后使用真空抽吸由喷嘴拾取这些球体，并使用3D生物打印机在针阵列上组装。然后允许球体在针阵列上熔断。三维生物印迹三天后，球状体作为完整的贴片被去除，已经自发地发生跳动。 3D生物印迹的心脏贴片显示组件球体的机械整合，并且在心脏组织再生和心脏病的3D模型中是非常有希望的。

在步骤4.4（共培养）结束时，每个孔中的细胞应在超低附着96孔U底板的底部聚集，通过重力形成球体。这些球体包含hiPSC-CM，HCF和HUVEC，并且可以在光学显微镜下目视检查，其中它们应该通过二维投影呈现圆形（ 图1 ）。在步骤6.3结束时，3D生物印迹的心脏贴片应该包含组织空隙，这是由于针阵列产生的针孔（ 图2左侧）。在这?...

Watch this Scientific Journal Video about Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting at JoVE.com

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

该协议描述了不使用生物材料的心脏组织的3D生物印迹。 3D生物印迹的心脏贴片显示组件球体的机械整合，并且在心脏组织再生和心脏病的3D模型中是非常有希望的。

使用3D生物印刷创建心脏组织展示球体的机械整合

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

creation-cardiac-tissue-exhibiting-mechanical-integration-spheroids

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.9K 次观看.  Johns Hopkins Hospital. 该协议描述了不使用生物材料的心脏组织的3D生物印迹。 3D生物印迹的心脏贴片显示组件球体的机械整合，并且在心脏组织再生和心脏病的3D模型中是非常有希望的。 

视频: Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

等离子体表面图案光刻技术的蜂窝网络创作

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

科研

生物工程

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

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 ...

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

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

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 ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

基于网模的无支架三维心脏组织创建方法

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

明胶美沙克里洛醇水凝胶生物油墨3D生物印刷方案

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

在三维生物打印中使用多层水凝胶生物油墨，实现均匀细胞分布

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

将3D多细胞乳房球体直接生物打印到内皮网络

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

使用人iPSC衍生的心肌细胞，心脏成纤维细胞和内皮细胞制造3D心脏微组织阵列

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

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 ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

用于工程神经样组织的小鼠皮质星形胶质细胞的3D生物打印

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...