作者承认以下资金来源: 为心血管研究提供资金的魔力。

1. 心肌细胞的制备
<ol><li>用基底膜基质和培养人诱导的多能干细胞 (hiPSCs) 6 井板, 如前所述的17。</li>
	<li>使用以前描述的方法18将 hiPSCs 区分为 hiPSC CMs。</li>
	<li>在 16-18 d 的分化后, 通过冲洗每个井与2毫升的1x 磷酸盐缓冲盐水 (PBS), 不含钙或镁, 然后孵化与1毫升/井的胰蛋白酶或细胞离解试剂 (见表材料) 室温下5分钟。</li>
	<li>与胰蛋白酶或细胞离解试剂 (参见材料表) 使用同样数量的罗斯威尔公园纪念协会 (RPMI) 细胞媒介补充与 B-27 (RPMI/B-27 细胞媒介)。吸管向上和向下松开任何粘附细胞。</li>
	<li>用10毫升血清学吸管收集悬浮心肌细胞, 将其转移到50毫升圆锥管。</li>
	<li>离心细胞悬浮在 250 x g 5 分钟室温下获得细胞颗粒。</li>
	<li>并用重悬10毫升 RPMI/B-27 细胞培养基中的颗粒。</li>
	<li>将20µL 的细胞悬架与同等数量的0.4% 台盼蓝溶液结合, 轻轻搅拌。</li>
	<li>使用手动 hemocytometer 计数和获得细胞悬浮液的浓度和细胞活性。</li>
</ol>2. 成纤维细胞的制备
<ol><li>启动一个人心脏成纤维细胞 (HCF) (成人心室类型) 单元格线的培养, 如前所述16。</li>
	<li>在室温16下, 用适当数量的胰蛋白酶或细胞离解试剂 (见材料表) 将 HCFs 悬浮在5分钟内。对于 T175 瓶, 使用10毫升的胰蛋白酶或细胞离解试剂。</li>
	<li>用同等体积的培养基中和胰蛋白酶或细胞离解试剂 (见材料表), 然后将样品转移到50毫升锥形管中, 离心机在室温下以 250 x g为5分钟的细胞悬浮液, 以获得颗粒。</li>
	<li>并用重悬10毫升成纤维细胞生长介质中的颗粒 (见材料表)。</li>
	<li>将 HCF 细胞悬浮液的20µL 与同等量的0.4% 台盼蓝溶液结合, 轻轻搅拌。</li>
	<li>使用自动单元格计数器或手动 hemocytometer 计数并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>启动人类脐静脉内皮细胞 (HUVEC) 线的培养, 如前所述17。在室温17下, 用适当数量的胰蛋白酶或细胞离解试剂 (见材料表) 将血管内皮细胞悬浮在3分钟内。中和胰蛋白酶或细胞离解试剂 (见材料表) 使用同等体积的介质, 然后转移到一个50毫升圆锥管和离心机的细胞悬浮在 250 x g 5 分钟在室温下获得颗粒。 注: 对于 T175 瓶, 使用10毫升的胰蛋白酶或细胞离解试剂。</li>
	<li>并用重悬10毫升内皮细胞生长培养基中的颗粒 (见材料表)。</li>
	<li>结合20µL 的 HUVEC 悬浮液, 并将其与同等数量的0.4% 台盼蓝溶液进行染色, 并轻轻搅拌。</li>
	<li>使用自动单元格计数器或手动 hemocytometer 计数和获得细胞悬浮液的浓度和细胞活力。</li>
</ol>4. 创建吊滴球体
<ol><li>将包含850孔 (每个直径350µm) 的悬挂放置装置放入无菌6井板中。</li>
	<li>隔离上述三种类型的细胞: hiPSC-CMs、HCFs 和血管内皮细胞。将它们与 70% iPSC CMs、15% HCFs 和15% 血管内皮细胞的比例结合在一个50毫升圆锥管中, RPMI/B-27 细胞介质的浓度为每毫升247.5万个细胞。</li>
	<li>将4毫升的细胞悬浮液 (每毫升247.5万个细胞) 分配给一个超低附着物悬挂系统的每一个井 (其中微孔直径为350µm), 坐在6井板上。</li>
	<li>球体将自发地在12小时内将细胞悬浮液放入吊滴装置中。继续养殖为共 72 h (3 d) 在37°c, 5% CO2, 和95% 湿气为成熟的球体。</li>
	<li>经过72小时的养殖, 通过将设备放入介质中, 并轻轻旋转, 将球体从悬挂装置中释放出来, 在悬挂系统底部的毛孔中收获球体。</li>
</ol>5. 使用新颖的网模创建3维补丁
<ol><li>将底座、底板、底网和10层侧网组装起来, 用一副无菌钳来制造新型模具。首先, 使用拐角柱堆叠并对齐底座、底方板和底部网。然后, 堆叠和层10侧网在交替的方向, 以创建一个良好的不锈钢齿网。</li>
	<li>用 PBS 或介质冲洗填充底座, 以减少表面张力, 然后将装配模转移到填充底座上。</li>
	<li>用同一方法将网模腔与 PBS 或介质一起湿。将球体慢慢填入所需尺寸的预设腔体 (2 x 2 x 1 毫米, 4 x 4 x 1 毫米, 或 6 x 6 x 1 毫米)。 注: 确保球体的预期体积的120% 被送入腔内, 使细胞聚集紧密连接并保持静止。</li>
	<li>将顶部网和顶方板装配到拐角的柱子上, 并确保塞子和保温管的安全, 防止球体的冲刷。</li>
	<li>将填充的网模系统转移到6井板上, 6 毫升的 RPMI/B-27 细胞介质, 或进入一个无菌容器, 有足够的介质覆盖整个组装模具。</li>
	<li>在 7-10 天, 在 3000-4000 x g上, 在摆动的振动筛上孵化6井板, 并装满模具系统。 注意: 孵化的持续时间取决于心脏斑块的大小。随着斑块的增大, 心脏斑块成熟时需要增加潜伏期。</li>
</ol>6. 从新型网模中去除修补程序
<ol><li>将模具系统从介质中取出, 并将其放在无菌的10厘米盘中灭菌处理垫上。</li>
	<li>卸下保温管、塞子、顶方板和顶网。小心地滑出分层的侧网一个一个用一对消毒钳。全麻拔管后, 在底部网上获得完整的心脏修补。</li>
	<li>用无菌钳把底网接上, 用5毫升的 RPMI/B27 介质将底网转移到35毫米的盘子里。在盘子里慢慢地旋转网, 或者用不育的细胞刮刀轻轻地松开底部网的补丁。从底网分离后, 可以用 RPMI/B27 培养基培养心脏组织。</li>
	<li>继续在35毫米的菜肴中孵化游离漂浮的3维心脏补丁。功能同步跳动可以观察早在24小时后, 从模具去除。</li>
	<li>从网模中去除修补后, 观察其形状保持完整性, 同步跳动强度随时间增加。 注: 3 维网状模心片在全麻拔管后表现为 cardiospheres 元件的电集成。我们观察到每分钟60到80次的跳动频率, 持续时间超过60天。</li>
</ol>

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作者没有什么可透露的。

该方法的意义在于其重现性和结果多层心肌组织的有效性。在心脏组织工程领域, 目前的目标之一是确定一种方法来构建跳动, 多层和功能3维心脏补丁。我们报告了一个有效的和可重复的方法创建多层心脏组织的直接人工播种的球体, 由心肌细胞, 内皮干细胞, 成纤维形成一个新的网霉菌。本方法所用的网模有各种不同的尺寸, 可用于从 2 x 2 x 1 毫米到 6 x 6 x 1 毫米不等的组织的创建。我们所描述的技术也可以根据所需的组织几何对不同的模具形状和系统进行修改。所描述的细胞浓度和比值被优化以前的 hiPSC CMs, 和对不同的细胞类型的修改将需要额外的评估。
利用 bioprinter 技术对3维心脏组织的体外创造进行了探索, 希望能建立治疗和诊断模型。然而, 目前的生物打印方法是繁琐的, 需要大量的时间和使用辅助支撑结构, 如针或脚手架材料。这些辅助结构的存在减少了营养素和氧气的扩散18。因此, 中央坏死是目前3维心脏结构的主要关注, 由于缺乏毛细血管提供氧气和营养素19。坂口等应用夹定向细胞片的方法, 利用血管床生物反应器开发内皮网络20。在这里提出的网模系统中, 模腔可以允许足够的养分和氧气的扩散, 而不需要任何支撑结构或支架。此外, net 模具的方法是有效的, 不需要专门的技能, 球体播种的模具和维护的后续补丁。因此, 它是一个快速和廉价的方式获得同步跳动和功能性心脏补丁。
为了优化各悬滴装置的球体直径和球体数量, 对细胞类型和数量进行了故障排除和修正。对于这种基于模塑的方法, 球形直径和球体的数量对于创建一个足够大小的功能性心脏组织至关重要。我们尝试不同的细胞浓度, 以优化最佳球体大小为心脏组织创造。三种类型的细胞通过悬挂装置产生心脏球体, 其中包括 70% iPSC CMs、15% HCFs 和15% 血管内皮细胞。在光镜下, 在每微孔中自发形成的球体在72小时后容易被观察到跳动。我们观察到, 球体的直径随着吊滴装置的细胞浓度而增加。我们尝试了不同浓度的球体, 从100球体到300球体在每个设备, 4 毫升的 RPMI/B27 培养基。经过多次试验, 我们发现每毫升247.5万细胞的浓度产生了球体的最佳直径。随着球体直径的优化, 我们在堆积网模中对球体进行了轻松的采集和播种, 是建立多层心脏组织的网状模化方法的重要步骤。
该协议最关键的步骤是球体大小的优化。值得一提的是, 这种方法所使用的球体比其他生物打印技术中使用的球形体的大小小21。由于营养和氧22的扩散不足, 较大的球体已被发现有中心坏死。随着球体直径较小, 足够的养分和氧气扩散到每个球体的中心更容易完成, 增加细胞的生存能力在所有区域的结果补丁。在这里提出的协议中, 球状直径为350µm 允许改善细胞的生存能力和更多的接触表面积之间的球体, 我们相信有助于成功创建的心脏组织使用这个网霉菌。因此, 这是所述方法中的一个关键步骤。为了适应其他细胞类型的协议, 需要额外的球体尺寸优化实验。
这种方法的局限性包括无法精确控制球体的堆叠。这就导致了不均匀的区域产生的组织补丁和异质厚度。此外, 虽然实际动手时间为球体的播种是极小的, 创建大型多层补丁需要一个延长的文化时间, 以允许融合的球体。
对于未来的应用, 这种新型的基于网模的方法是一种有效的、可重现的方法, 用于创建3维、无支架的心脏组织, 用最少的人工努力。所产生的多层组织包括融合球体, 结构完整性良好, 同步跳动行为。这种基于模塑的方法提出了一种经济高效和可伸缩的替代现有的工程组织的生物制造方法, 并有潜力创造临床适用的心脏组织治疗心力衰竭的目的是取代缺血性或功能失调的心肌细胞23。此外, 这种方法可以用来创建心脏组织为病人特定的筛查潜在的新药物治疗24。

目前心脏组织工程的目的是开发一种替代或修复损伤心肌组织的结构和功能的治疗方法1。方法建立3维心脏组织模型, 显示本机心脏组织的重要收缩和电生理特性, 迅速扩大2,3。在研究4、5中探索和使用了各种战略。这些方法包括使用特定的合成和天然生物活性水凝胶, 如明胶, 胶原蛋白, 纤维蛋白和肽6, 生物墨水沉积技术2和生物打印技术7。
研究表明, 无支架的方法可以产生可比的组织作为生物材料的方法, 而不存在的缺点, 纳入外国脚手架材料8。奥伦 Caspi等人证明, 结合各种类型的细胞, 使生成高血管化的人类工程心脏组织9。下巴等人开发了一种3维印刷方法的心脏补丁创建从球体。所产生的斑块由心肌细胞、纤维细胞和内皮干细胞组成, 比例为 70:15:15.10。球体已被证明是有效的 "积木" 的无支架的心脏组织创建, 因为他们耐缺氧, 并拥有足够的机械完整性, 植入11,12。以前的研究已经展示了几种制造球体的方法, 包括使用吊滴法、微调烧瓶13、微流控系统14和非粘附的培养表面, 不涂或涂有琼脂糖微模15。在本协议中, 我们使用悬挂放置装置, 其中包含孔, 以凝聚数以百计的球体一次。
本研究提出了一种新的、高效的无支架的心脏组织创建方法, 包括将球体人工播种到方形模腔中, 并在振动筛上孵化组织以进行成熟。在通常的静态培养条件下, 氧扩散仅限于组织结构的外部, 导致中心坏死。然而, 随着网模, 所有的球体种子进入模具都沉浸在介质中, 不断的射流运动, 使养分和氧气的扩散增加。此外, 这种基于模具的方法允许同时创建不同大小的组织补丁, 用最少的人工努力, 由此产生的组织可以很容易地从模具中删除。这种新的方法允许有效和可重复的创建无支架, 多层心脏补丁。

<table border="0" cellpadding="0" cellspacing="0" style="width:708px;" width="708">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Human Cardiac fibroblasts (HCF)</td>
			<td style="width:139px;">Sciencell</td>
			<td style="width:119px;">6310</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">FM-2 Consists of Basal Medium</td>
			<td style="width:139px;">Sciencell</td>
			<td style="width:119px;">2331</td>
			<td style="width:220px;">HCF culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Human umbilical vein endothelial cells (HUVEC)</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:119px;">CC-2935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">EGM+Bullet Kit&#160;</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:119px;">CC5035</td>
			<td>HUVEC culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">E8 media&#160;</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">A1517001</td>
			<td>HiPSC culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Geltrex&#160;</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">A1413202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">TrypLE Express Enzyme (1X)</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">12604013</td>
			<td>Trypsin and Cell dissociation reagent</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">RPMI media</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">11875093</td>
			<td style="width:220px;">RPMI media with B-27 supplement is hiPSC-CM culture medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">B-27 supplement (50x)</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">17504044</td>
			<td style="width:220px;">RPMI media with B-27 supplement is hiPSC-CM culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Novel net mold&#160;</td>
			<td style="width:139px;">TissueByNet Co.,Ltd</td>
			<td style="width:119px;">NM25-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hanging drop plate</td>
			<td style="width:139px;">Kuraray Co.,Ltd</td>
			<td style="width:119px;">MPc350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">6 well plates&#160;</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:119px;">CLS-3516</td>
			<td></td>
		</tr>
	</tbody>
</table>

a net mold-based method of scaffold-free three-dimensional cardiac tissue creation

该协议描述了一种新的和简单的基于网络模型的方法, 以创建三维 (3 维) 心脏组织没有额外的支架材料。人诱导的多潜能干细胞衍生心肌细胞 (iPSC cms), 人心脏成纤维细胞 (HCFs) 和人脐静脉内皮干细胞 (血管内皮细胞) 被隔离, 并用于产生细胞悬浮与 70% iPSC CMs, 15% HCFs, 15% 血管内皮细胞。他们是共同培养的超低附件 "悬挂下降" 系统, 其中包含孔, 以凝聚数以百计的球体一次。在共培养3天后, 细胞聚集并自发形成跳动球体。球体的收获, 种子成一个新的模具腔, 并培养在一个振动筛在孵化器。球体成为成熟的功能组织大约7天后播种。所合成的多层组织包括融合球体, 结构完整性良好, 同步跳动行为。这种新方法具有很好的潜力, 可以作为一种重现性和成本效益高的方法, 为将来的心力衰竭治疗创造出工程组织。

在我们的实验中, 我们利用细胞悬浮 70% iPSC CMs, 15% HCFs, 15% 血管内皮细胞在 RPMI/B-27 细胞介质中浓度247.5万细胞每毫升。在建立细胞悬浮液后, 我们将4毫升的细胞悬浮液分配给超低附着悬挂系统的每个井, 如协议步骤4.3 所述。在3天的文化在37°c、5% CO2和95% 湿气以后, 使用垂悬的下落系统导致了数以百计的跳动的球体的自发形成。球体很容易被观察到在光显微镜下跳动4X 和10X 放大, 平均直径为350µm 后72小时的文化 (图 1)。
在该协议步骤6.5 的末尾, 在收获球体后, 该模具很容易被简单的吹打方法播种。霉菌的培养允许球体的融合;随后去除霉菌导致一个完整的心脏补丁与机械完整性。从霉菌和 24 h 的培养后, 观察到心脏组织的功能性和同步跳动, 确认球体的机械整合 (图 2, 左)。我们还观察到, 多层球体在光镜下以协调的方式自发跳动 (图 2, 右)。
采用心脏标记肌钙蛋白 t 共聚焦成像技术对3维心片进行了免疫组化分析, 结果表明, 排列好的心肌细胞均表现为肌钙蛋白 T 表达 (图 3)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58252/58252fig1.jpg"> 图 1: 创建吊滴球体.人诱导的多潜能干细胞衍生心肌细胞 (hiPSC cms), 人心脏成纤维细胞 (HCFs) 和人脐静脉内皮干细胞 (血管内皮细胞) 被隔离, 并用于产生细胞悬浮与 70% iPSC CMs, 15% HCFs, 15% 血管内皮细胞, 在每毫升247.5万细胞浓度。72小时后, 在光镜下容易地观察到每个微孔中自发形成的球体。左面板中的刻度条 = 1000 µm;右面板中的刻度条 = 400 µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58252/58252fig1large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58252/58252fig2.jpg"> 图 2: 建立一个多层心肌组织, 显示组件球体的整合.从净霉菌分离后, 3 维心脏修补器保持其形状, 并显示同步跳动, 表明球体的机械集成, 如左面板 (其中刻度条 = 1000 µm) 所示。球体的堆积多层膜经光镜检查, 如右面板 (其中鳞片杆 = 400 µm) 所示, 并观察到自发跳动。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58252/58252fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58252/58252fig3.jpg"> 图 3: 对心脏组织的免疫荧光评价.在免疫荧光分析中, 发现基于3维的网状心肌组织由心肌肌钙蛋白 T 表达的排列良好的细胞组成。刻度条 = 50 µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58252/58252fig3large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>

Watch this Scientific Journal Video about A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation at JoVE.com

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

基于网模的无支架三维心脏组织创建方法

本协议描述了一种基于净模的方法来创建三维无支架的心脏组织, 结构完整性和同步跳动行为令人满意。

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

a-net-mold-based-method-scaffold-free-three-dimensional-cardiac

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.8K 次观看.  The First Hospital of Jilin University. 本协议描述了一种基于净模的方法来创建三维无支架的心脏组织, 结构完整性和同步跳动行为令人满意。

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

平面和立体印刷的导电油墨

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

人体软骨组织制作采用三维喷墨打印技术

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

倒胶体晶体聚（乙二醇）脚手架制作:肝脏组织工程三维细胞培养平台

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

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

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

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 ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

三维肽支架中哺乳动物细胞的培养

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

原发性肾组织段的显微解剖与新型无支架结构技术的结合

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...