本研究由宁波市自然科学基金（2022J121、2023J159）、宁波市自然科学基金重点项目（2021J256）、复旦大学高分子工程国家重点实验室开放基金（K2024-35）和浙江省动脉粥样硬化疾病精准医学重点实验室（2022E10026）资助。感谢宁波大学健康科学中心核心设施的技术支持。

1. κ -角叉菜胶亚微凝胶混悬浴的制备
<ol><li>通过将 1.75 g κ-卡拉胶粉末添加到 1,000 mL 玻璃瓶中的 500 mL 磷酸盐缓冲盐水（PBS，pH 7.4）溶液中，制备 500 mL κ-卡拉胶悬浮浴（0.35% wt/vol）。</li>
	<li>将 70 mm 磁力搅拌棒引入玻璃瓶中以搅拌水性混合物。拧紧玻璃瓶盖，然后松开半圈。</li>
	<li>将玻璃瓶置于 70 °C 水浴中并加热。以 300 rpm 的速度打开磁力搅拌器，放置瓶子，搅拌至聚合物完全溶解。</li>
	<li>取另一个玻璃瓶，将其放入高压灭菌器中进行灭菌，在 121 °C 下运行 30 分钟。</li>
	<li>高压灭菌器冷却至 80 °C 后，从灭菌器中取出瓶子，将其转移到超净工作台上进行进一步处理。</li>
	<li>使用 0.22 μm 过滤器将完全溶解的 κ-角叉菜胶溶液过滤到灭菌的玻璃瓶中。 注：当溶液很热时，应迅速进行过滤，以防止 κ-角叉菜胶冷却并阻塞过滤器。</li>
	<li>将 κ-角叉菜胶溶液储存在 4 °C 以诱导阳离子交联凝胶 12 小时。</li>
	<li>在 25 °C 下，使用 60 mm 磁力搅拌器以 1200 rpm 的搅拌速度机械研磨 κ-角叉菜胶水凝胶，直至其成功转变为液态，大约需要 60 分钟。 注意：将 κ-卡拉胶水凝胶储存在 25-37 °C 之间，以避免在低温下凝胶化或在高温下溶解。</li>
</ol>2. 甲基丙烯酸明胶/丝素蛋白甲基丙烯酸酯复合生物墨水的制备
<ol><li>通过在 PBS 溶液 （pH 7.4） 中混合 10% （wt/vol） 甲基丙烯酸明胶 （GelMA） 和 6% （wt/vol） 丝素蛋白甲基丙烯酸酯 （SFMA） 来制备复合生物墨水。分别称取 2.0 g GelMA 和 1.2 g SFMA 粉末。</li>
	<li>将粉末缓慢加入两个 50 mL 离心管中。将 10 mL PBS 溶液分别添加到 50 mL 离心管中。</li>
	<li>向每个容器中加入 10 mm 磁力搅拌器，并在 45 °C 的水浴中不断搅拌和加热。 让 GelMA 和 SFMA 粉末完全溶解，大约需要 30 分钟。</li>
	<li>在 45 °C 下连续搅拌，将 GelMA 和 SFMA 溶液以等体积混合。 使用 100 μm 孔径过滤器过滤复合生物墨水。</li>
	<li>在生物安全柜中通过紫外线照射对复合 GelMA/SFMA 生物墨水消毒 12 小时。 注意：立即准备并使用生物墨水，以防止 SFMA 因自组装而过早凝胶化。</li>
</ol>3. κ -角叉菜胶亚微凝胶悬浮浴的流变学特性
<ol><li>启动并准备流变学相关设备。<ol><li>打开空气压缩机 30 分钟，确保压力达到 30 psi。逆时针转动拉杆，拆下轴承夹。固定下面的黑色盖子，然后顺时针旋转仪器顶部的旋钮轴。</li>
		<li>打开流变仪电源并使其初始化。打开循环水开关，让温度达到 15 °C。</li>
		<li>打开流变仪控制软件以建立在线连接。</li>
	</ol></li>
	<li>校准流变仪并安装平行板几何形状。<ol><li>在控制软件中进行仪器校准，主要涉及调整校准仪器惯量路径。将直径为 40 mm 的平行板几何形状安装到拉杆的末端。按住 Lock 按钮 3 秒，将电机轴移动到初始位置，以实现一致的几何图形放置。</li>
		<li>在控制软件中选择 校准管理器 ，然后单击 惯性、摩擦校准 和 旋转映射 以校准平行板几何结构。</li>
	</ol></li>
	<li>通过执行标准间隙归零程序将流变仪间隙高度归零，以确保精确测量。</li>
	<li>设置实验步骤，包括 0.01 至 1000 1/s 剪切速率的流梯度、10 rad/s 时 0.1% 至 100% 应变的振幅扫描、持续 60 s 的多级振荡时间扫描，在 10 rad/s 时交替应变为 1% 和 100%，以及持续 120 s 的多级峰值保持扫描，交替剪切速率为 0.1 1/s 和 10 1/s。</li>
	<li>将 2 mL 0.35% κ-卡拉胶悬浮液滴到流变仪的 Peltier 板上。</li>
	<li>将几何间隙定位到 510 μm，并去除夹紧装置边缘的任何多余溢流。将样品间隙设置为 500 μm，以允许样品略微超出夹具边缘。</li>
	<li>通过从实验设计选项中选择 Flow Ramp 来执行流动实验。<ol><li>在试验设计中选择 Flow Ramp 检验。在 25 °C 的温度下，将剪切速率设置为 0.001 至 10 1/s。</li>
		<li>对添加的样品执行步骤 3.5 和 Flow Ramp 实验。</li>
	</ol></li>
	<li>执行循环峰值保持测试以评估材料的回收性能。<ol><li>在实验设计中选择 Peak Hold sweep 并在 25 °C 下设置 5 个连续实验，时间为 120 s。</li>
		<li>将第一、第三和第五步的剪切速率设置为 0.01 1/s，将第二和第四步的剪切速率设置为 10 1/s。</li>
		<li>添加 2 mL 的新样品。执行步骤 3.5 和循环恢复测试。</li>
	</ol></li>
	<li>进行粘弹性分析以评估 κ-角叉菜胶悬浮浴的潜在凝胶-溶胶转变。<ol><li>在实验设计中选择 Amplitude Sweep 检验。在 25 °C 的温度下将振荡应变设置为 0.1% 至 100%。 单击 Start 对添加的样品执行 Amplitude Sweep 实验。</li>
	</ol></li>
	<li>执行替代应变分析以评估材料的弹性恢复性能。<ol><li>在实验设计中选择 Oscillation Time Sweep 并在 25 °C 下设置 5 个连续实验，时间为 60 s。</li>
		<li>将第一、第三和第五步的应变设置为 1%，第二步和第四步的应变设置为 100%。</li>
		<li>添加 2 mL 的新样品。执行步骤 3.5 和连续循环恢复测试。 注：每次实验测试后清洁几何板和 Peltier 板，并添加 2 mL 的新样品。</li>
	</ol></li>
</ol>4. 使用定制设计的微挤压生物打印机打印仿生水凝胶支架
<ol><li>使用 3D 图形软件设计 STL 格式的立方体，并从 3D 数据库下载组织状模型 （www.thingiverse.com; https://3d. nih.gov/.）。<ol><li>将 STL 格式模型导入 PANGO 软件并输入打印参数。设置模型在 3D 空间中的具体 X、Y、Z 坐标，输入模型期望的缩放尺寸（以毫米 （mm） 为单位），调整物体在 X、Y、Z 轴上的尺寸和缩放比例，根据需要调整模型在空间中的旋转角度。</li>
		<li>将估计的细丝直径（大多数生物墨水通常约为 200-500 μm）输入到层厚字段中，以确定每层的 Z 轴厚度。根据预期的线条粗细（约 10-90 mm/s）设置打印速度，并将填充密度设置为 30%-80%。</li>
		<li>将最终参数作为 G 代码导出到 SD 卡上。</li>
	</ol></li>
	<li>运行生物打印机和微挤出泵。<ol><li>打开微挤出泵控制器，选择相应体积的注射器 （5 mL），并将挤出速率设置为 0.08 mL/min，并将挤出持续时间设置为与通过将水凝胶体积除以挤出速率获得的所需打印时间相协调。</li>
		<li>将内径为 210 μm 的针头注射器安装到生物打印机的注射器槽中。</li>
		<li>调整注射器控制器，确保注射器的柱塞与螺钉紧密接触。</li>
		<li>将 3 mL 悬浮浴放入 35 mm 细胞培养皿中。根据代码设置将培养皿放置在平台上，并确认打印头高于培养皿底部 1 毫米。 注意：进行预测试以确保打印过程中打印平台上培养皿位置的准确性。</li>
		<li>将包含代码的 SD 卡插入 3D 生物打印机，激活代码文件，启动生物打印机，然后单击控制器上的 “开始 ”按钮。</li>
	</ol></li>
	<li>结构的加工<ol><li>将打印的构建体暴露在 405 nm 蓝光下 1 分钟以引发光交联。</li>
		<li>使用 1 mL 移液管去除 κ-角叉菜胶凝胶，然后加入 3 mL PBS （pH 7.4） 进行洗涤和随后去除。重复此过程以彻底清洗。 注意：轻柔地执行洗涤过程，以避免损坏打印的结构。</li>
	</ol></li>
</ol>5. 食管肌肉层类似物的嵌入式 3D 生物打印
<ol><li>培养兔食管平滑肌细胞 （eSMC）。<ol><li>使用 15 mL 补充有 10% 胎牛血清和 1% 青霉素/链霉素的 DMEM 培养基，在 2 x 106 个细胞的 T75 培养瓶中开始培养。</li>
		<li>将 eSMC 保持在 37 °C，在 5% CO2 和 95% 空气的潮湿环境中，直到它们达到 80% 汇合。</li>
	</ol></li>
	<li>食管肌层生物墨水的制备<ol><li>达到汇合后吸出培养基，并用 5 mL 的 1x PBS 洗涤 eSMC。</li>
		<li>向培养瓶中加入 2 mL 0.2% 胰蛋白酶-EDTA，孵育 2 分钟以消化细胞。点击烧瓶以将细胞从墙上分离。</li>
		<li>通过添加 2 mL DMEM 终止消化，并将细胞悬液转移到 15 mL 离心管中。使用含有 10 μL 细胞悬液的细胞计数器测量细胞密度。</li>
		<li>以 675 x g 离心 3 分钟，小心吸出上清液，并将细胞沉淀重悬于复合 GelMA/SFMA 生物墨水中（如步骤 2.1 中制备）。调整最终细胞浓度，使生物墨水中的密度达到 10 x 106 个细胞/mL，从而为后续的 3D 生物打印应用优化条件。</li>
	</ol></li>
	<li>3D 生物打印机和相关材料的准备<ol><li>在标准灭菌条件（121 °C 30 分钟）下高压灭菌必要的打印工具，包括注射器针头、镊子、磁力搅拌器和 1000 mL 硅酸盐玻璃瓶，以确保打印过程的无菌环境。</li>
		<li>将生物打印机放入生物安全柜中，并用紫外线 （UV） 消毒 30 分钟以保持无菌。</li>
		<li>准备 50 mL 无菌 κ-角叉菜胶和 5 mL 无菌 GelMA/SFMA 复合生物墨水，分别如步骤 1 和步骤 2 所述。</li>
		<li>设计模型，设置所需参数，并按照步骤 4.1 中的概述准备生物打印机。</li>
	</ol></li>
	<li>生物打印过程的启动<ol><li>执行步骤 4.2 和 4.3 中描述的程序以生物打印 3D 食管肌肉层，然后进行 PBS 洗涤和更换。</li>
		<li>在整个打印过程中使用无菌技术进行细胞打印，以最大限度地降低打印结构中细胞污染的风险。</li>
	</ol></li>
	<li>打印后细胞培养基更换<ol><li>小心吸出 PBS，并用 3 mL 补充有 10% 胎牛血清和 1% 青霉素/链霉素的 DMEM 代替。确保打印的构建体完全浸没以提供最佳的细胞生长条件。</li>
		<li>将含有打印构建体的细胞培养皿在设置为 37 °C 和 5% CO2 的培养箱中孵育。每天用新鲜培养基更换细胞培养基，每次使用 3 mL，并使用光学显微镜监测构建体内细胞的状态。</li>
	</ol></li>
</ol>6. 通过 Live/Dead 测定染色评估打印构建体中 eSMCs 的活力
<ol><li>孵育 5 小时后取出细胞培养基，并用 PBS 洗涤载有细胞的构建体。</li>
	<li>将 2 mL 标准钙黄绿素-AM/碘化丙啶溶液 （1：1000） 添加到载有细胞的打印构建体中并孵育。</li>
	<li>孵育 45 分钟后，用 PBS 洗涤构建体 3 次，然后加入新鲜的细胞培养基。</li>
	<li>使用共聚焦激光扫描显微镜 （CLSM） 观察活细胞和死细胞并捕获荧光图像。</li>
</ol>7. 通过 FITC-鬼笔环肽/DAPI 染色观察打印构建体内的 eSMC
<ol><li>使用异硫氰酸荧光素 （FITC） 标记的鬼笔环肽（绿色）对打印的构建体进行染色，用于 F-肌动蛋白和 4'，6-二脒基-2-苯基吲哚（DAPI，蓝色），用于孵育 5 天后的核。</li>
	<li>固定液制备<ol><li>将 4 mL 1x PBS 与 0.16 mL 多聚甲醛 （PFA） 混合在 5 mL 聚乙烯 （PE） 管中，达到 4% 的最终浓度。</li>
		<li>将含有生物打印构建体的培养皿放在干净的表面上。去除培养基并用 PFA 溶液轻轻覆盖构建体，确保其完全浸入水中。在丢弃溶液之前，让固定在室温下进行 20 分钟。 注：在添加和去除过程中小心处理液体，以保持生物打印结构的结构完整性。</li>
		<li>将 2.5 mL 的 1x PBS 引入培养皿中，将构建体完全浸没以去除任何残留的染色试剂。</li>
	</ol></li>
	<li>细胞膜透化<ol><li>通过在 5 mL PE 管中将 20 μL Triton X-100 添加到 4 mL 1x PBS 中，配制 0.5% Triton X-100 溶液。</li>
		<li>在室温下将 Triton X-100 溶液施用到构建体中 30 分钟以渗透细胞膜，然后去除溶液。</li>
		<li>将 2.5 mL 的 1x PBS 引入培养皿中，将构建体完全浸没以去除任何残留的染色试剂。</li>
	</ol></li>
	<li>阻断非特异性结合<ol><li>通过将 0.12 mL BSA 添加到 4 mL 1x PBS 中，在 5 mL PE 管中制备 3% 牛血清白蛋白 （BSA） 封闭溶液。</li>
		<li>在室温下将 BSA 溶液引入构建体 30 分钟以阻断非特异性位点，然后丢弃溶液。</li>
	</ol></li>
	<li>用 FITC-鬼笔环肽进行肌动蛋白染色<ol><li>在 5 mL PE 管中制备 0.2% FITC-鬼笔环肽溶液，加入 4 mL 1x PBS 和 8 μL 染色试剂。</li>
		<li>将构建体在室温下避光浸入该溶液中 60 分钟，然后丢弃溶液。</li>
		<li>向培养皿中加入 2.5 mL 的 1x PBS，将构建体完全浸没以去除残留的染色试剂。</li>
	</ol></li>
	<li>使用 DAPI 进行细胞核染色<ol><li>将 4 mL DAPI 染色溶液涂在构建体上，确保完全覆盖。</li>
		<li>在室温下避光孵育 20 分钟，然后去除 DAPI 溶液。</li>
	</ol></li>
	<li>最终洗涤和 PBS 浸没：向培养皿中加入 2.5 mL 的 1x PBS，完全浸没构建体以去除残留的染色试剂。 注意：提前准备染色溶液并轻轻处理。细胞在任何时候都不应在培养容器内进行干燥。</li>
	<li>使用共聚焦显微镜观察：使用共聚焦显微镜检查染色的构建体并对其进行成像，以评估细胞生长，确保全面记录结果。</li>
</ol>8. 使用 CCK-8 测定评估打印构建体内 eSMCs 增殖
<ol><li>在第 7 天和第 14 天从构建体中取出培养基。用 PBS 洗涤构建体以去除残留培养基和分离的细胞。</li>
	<li>按照制造商的说明将 CCK-8 溶液添加到每个构建体中。确保 CCK-8 溶液的体积足以完全覆盖每个构建体。</li>
	<li>将构建体与 CCK-8 溶液在 37 °C 下孵育 2 小时。孵育后，小心地将上清液转移到新的 96 孔板中。</li>
	<li>使用酶标仪测量 450 nm 处的吸光度。</li>
</ol>

使用 κ-角叉菜胶亚微凝胶培养基推进 3D 生物打印

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=durability,+contractility+and+vascular+network+formation+in+3d+bioprinted+cardiac+endothelial+cells+using+alginate-gelatin+hydrogels.">durability, contractility and vascular network formation in 3d bioprinted cardiac endothelial cells using alginate-gelatin hydrogels.</a> Front Bioeng Biotech. 9, 63657 (2021).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+bioprinting+of+tough+hydrogel-based+vascular+conduits+for+functional+blood+vessels.">Microfluidic bioprinting of tough hydrogel-based vascular conduits for functional blood vessels.</a> Sci Adv. 8 (43), (2022).</li><li>Shao, L., Hou, R. X., Zhu, Y. B., Yao, Y. 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作者对本手稿中描述的产品没有经济利益。

用于生物打印的 κ-角叉菜胶亚微凝胶悬浮浴的制备是一个精心策划的过程，涉及几个关键步骤，以确保所得培养基表现出支持生物墨水所需的特性。最初，通过将 κ-卡拉胶粉末在高温下溶解在去离子水中，形成均匀的混合物来制备 κ-卡拉胶溶液。溶液的浓度是关键，必须针对后续的凝胶化进行优化。0.3-0.6% （wt/vol） κ-</stron...

组织工程支架，包括静电纺丝纤维、多孔海绵和聚合物水凝胶，通过提供支持细胞生长、组织再生和器官功能恢复的结构框架，在受损组织和器官的修复和重建中发挥关键作用 1,2,3.然而，传统支架在准确复制天然组织结构方面遇到了挑战，导致工程组织和天然组织不匹配。这种限制阻碍了缺陷组织的有效愈合，强调了支架设计进步以实现更准确的仿生学的迫切需求。三维 （3D） 生物打印是一种创新的制造技术，它使用生物材料墨水和细胞逐层精确构建复杂的生物组织结构4。在各种生物材料中，聚合物水凝胶成为理想的生物墨水，其独特的网络有助于细胞的原位封装，并关键地支持其生长 5,6。然而，当用作生物墨水时，许多柔软且高度水合的水凝胶在打印过程中往往会诱导打印的支架结构模糊或快速塌陷。为了应对这一挑战，嵌入式 3D 生物打印技术采用微凝胶浴作为支撑材料，可实现精确的软生物墨水沉积。水凝胶生物墨水凝胶化后，通过去除微凝胶浴获得具有复杂结构的精制仿生支架。明胶 7,8、琼脂糖9 和结冷胶10,11 等材料已被用于制造用于嵌入式 3D 生物打印的微凝胶浴，显着推动了软水凝胶在组织工程中的应用。然而，这些颗粒凝胶的微米级和不均匀粒径会对 3D 打印的分辨率和保真度产生不利影响 12,13,14。迫切需要制造一种具有小而均匀分散颗粒的凝胶状悬浮浮子，为实现高保真生物打印提供优势。
在该协议中，提出了一种具有均匀亚微米级的新型牺牲颗粒 κ-卡拉胶悬浮浴，用于嵌入式 3D 打印。这种快速干扰-去干扰过渡的创新亚微凝胶浴行为有助于精确制造具有高结构保真度的仿生水凝胶支架15。利用这种新的悬浮培养基，采用由甲基丙烯酸明胶和甲基丙烯酸丝纤维组成的复合生物墨水，成功打印了一系列具有多层组织结构的仿生组织和器官构建体。在这项研究中，我们选择食管作为 3D 生物打印仿生对象，主要是因为食管不仅具有多层组织结构，而且其肌肉层表现出内部圆形和外部纵向复杂的分层结构。确保这些层的正确对齐和组织对于功能性组织再生至关重要。因此，我们非常希望复制食道的多层结构。更重要的是，我们利用 κ-角叉菜胶亚微凝胶作为悬浮浴，使用 GelMA/SFMA 作为生物墨水来设计和构建用于组织工程的仿生支架。打印的食管可以通过反复磷酸盐缓冲盐水清洗轻松释放。此外， κ-角叉菜胶亚微凝胶浴不含细胞毒性物质，可确保高细胞相容性15。加载在各向异性支架内的平滑肌细胞表现出显着的扩散活性。这种均匀的亚微凝胶悬浮培养基为通过嵌入式 3D 生物打印制造复杂组织和器官提供了一条新途径。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D bioprinter</td>
			<td>Custom-designed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8217;,6-Diamidino-2-Phenylindole</td>
			<td>Solarbio Life Science</td>
			<td>C0065</td>
			<td>Ready-to-use</td>
		</tr>
		<tr>
			<td>405 nm UV light</td>
			<td>EFL</td>
			<td>XY-WJ01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counter</td>
			<td>Corning</td>
			<td>Cyto smart 6749</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica</td>
			<td>STELLARIS 5</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>VivaCell</td>
			<td>C3113-0500</td>
			<td>High Glucose, with Sodium Pyruvate and L-Glutamine</td>
		</tr>
		<tr>
			<td>Dynamic rotational rheometer</td>
			<td>TA Instrument</td>
			<td>Discovery HR-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Esophageal smooth muscle cells</td>
			<td>Supplied by the Department of Cell Biology and Regenerative Medicine, Health Science Center, Ningbo University</td>
			<td></td>
			<td>Primary cells from the rabbit esophagus</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>UE</td>
			<td>F9070L</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate labeled phalloidin</td>
			<td>Solarbio Life Science</td>
			<td>CA1610</td>
			<td>300T</td>
		</tr>
		<tr>
			<td>Gelatin methacrylate</td>
			<td>EFL</td>
			<td>EFL-GM-60</td>
			<td>60% substitution</td>
		</tr>
		<tr>
			<td>k-carrageenan</td>
			<td>Aladdin</td>
			<td>C121013-100g</td>
			<td>Reagent grade</td>
		</tr>
		<tr>
			<td>Lithium Phenyl (2,4,6-trimethylbenzoyl) phosphinate</td>
			<td>Aladdin</td>
			<td>L157759-1g</td>
			<td>365~405 nm</td>
		</tr>
		<tr>
			<td>Live-Dead kit</td>
			<td>beyotime</td>
			<td>C2015M</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Potenov</td>
			<td>PT-3502B</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Solarbio Life Science</td>
			<td>P1110&#160;</td>
			<td>4%</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Solarbio Life Science</td>
			<td>MA0110</td>
			<td>100 &#180;</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>VivaCell</td>
			<td>C3580-0500</td>
			<td>pH 7.2-7.4</td>
		</tr>
		<tr>
			<td>Silk fibroin methacrylate</td>
			<td>EFL</td>
			<td>EFL-SilMA-001</td>
			<td>39% substitution</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Solarbio Life Science</td>
			<td>T8200</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>VivaCell</td>
			<td>C100C1</td>
			<td>0.25%, without phenol red</td>
		</tr>
	</tbody>
</table>

embedded bioprinting of tissue-like structures using &#954;-carrageenan sub-microgel medium

利用颗粒水凝胶支撑浴的嵌入式三维 （3D） 生物打印已成为创建仿生支架的关键技术。然而，设计一种合适的凝胶悬浮培养基来平衡精确的生物墨水沉积与细胞活力和功能存在多重挑战，尤其是在实现所需的粘弹性方面。在这里，通过易于操作的机械研磨工艺制造了一种新型 κ-角叉菜胶凝胶支撑浴，产生均匀的亚微米级颗粒。这些亚微凝胶表现出典型的 Bingham 流动行为，具有较小的屈服应力和快速剪切稀化特性，有助于生物墨水的顺利沉积。此外， κ-卡拉胶微凝胶网络的可逆凝胶-溶胶转变和自愈能力确保了打印结构的结构完整性，从而能够创建具有明确结构特征的复杂、多层组织结构。打印后， κ-角叉菜胶亚微凝胶可以通过简单的磷酸盐缓冲盐水洗涤轻松去除。使用载细胞生物墨水的进一步生物打印表明，仿生构建体内的细胞具有 92% 的高活力，可快速扩展伪足，并保持稳健的增殖，表明这种生物打印策略在组织和器官制造方面的潜力。总之，这种新型 κ-角叉菜胶亚微凝胶培养基成为一种有前途的高质量嵌入式生物打印途径，对工程组织和器官的 体外 发育具有深远的影响。

Tissue engineering scaffolds, including electro-spun fibers, porous sponges, and polymer hydrogels, play a pivotal role in the repair and reconstruction of damaged tissues and organs by providing a structural framework supporting cell growth, tissue regeneration, and the restoration of organ function1,2,3. However, traditional scaffolds encounter challenges in accurately replicating native tissue structures, leading to a mismatch between the engineered and natural tissues. This limitation hinders the efficient healing of defective tissues, emphasizing the urgent need for scaffold design advancements to achieve more accurate biomimicry. Three-dimensional (3D) bioprinting is an innovative manufacturing technique that precisely constructs complex biological tissue structures layer by layer using biomaterial inks and cells4. Among various biomaterials, polymer hydrogels emerge as ideal bioinks with their distinctive network that facilitates in situ encapsulation of cells and crucially supports their growth5,6. Nevertheless, many soft and highly hydrated hydrogels tend to induce blurring or rapid collapse of printed scaffold structures during the printing process when used as bioinks. To address this challenge, embedded 3D bioprinting technology employs a microgel bath as a support material, allowing precise soft bioink deposition. Upon gelation of the hydrogel bioinks, refined bionic scaffolds with intricate structures are obtained by removing the microgel bath. Materials like gelatin7,8, agarose9, and gellan gum10,11 have been employed to create microgel baths for embedded 3D bioprinting, significantly advancing the application of soft hydrogels in tissue engineering. However, the micron-level and non-uniform particle size of these particulate gels detrimentally impacts the resolution and fidelity of 3D printing12,13,14. There is an urgent need to fabricate a gel-like suspension float with small and uniformly dispersed particles, offering advantages in achieving high-fidelity bioprinting.
In this protocol, a novel sacrificial granulate &#954;-carrageenan suspension bath with a uniform sub-micron level is presented for embedded 3D printing. This innovative sub-microgel bath behavior of rapid jamming-unjamming transition facilitates the precise fabrication of biomimetic hydrogel scaffolds with high structural fidelity15. Utilizing this new suspension medium, a series of biomimetic tissue and organ constructs featuring multi-layer tissue structures are successfully printed, employing a composite bioink composed of gelatin methacrylate and silk fibro methacrylate. In this study, we chose the esophagus as the 3D bioprinting biomimetic object mainly because the esophagus not only has a multi-layered tissue structure but also its muscle layer exhibits an internal circular and external longitudinal complex layering structure. Ensuring proper alignment and organization of these layers is essential for functional tissue regeneration. Therefore, we highly desire to replicate the multilayered architecture of the esophagus. More importantly, we utilized &#954;-carrageenan sub-microgels as the suspension bath and GelMA/SFMA as the bioink to design and construct a biomimetic scaffold for tissue engineering. The printed esophagus can be easily released by repeated phosphate-buffered saline washing. Moreover, the &#954;-carrageenan sub-microgel bath is free of cytotoxic substances, ensuring high cytocompatibility15. The smooth muscle cells loaded within anisotropic scaffolds exhibit a notable spreading activity. This uniform sub-microgel suspension medium offers a new avenue for the fabrication of complex tissues and organs through embedded 3D bioprinting.

作者聚焦：用于高分辨率 3D 生物打印的均相 κ-角叉菜胶亚微凝胶浴的开发

使用 κ-角叉菜胶亚微凝胶培养基对组织样结构进行嵌入式生物打印

3D bioprinting in a microgel-based suspension bath has provided a versatile platform for fabricating complex 3D structures using soft hydrogels, but it is still limited by the low print resolution and accuracy, hindering the practical applications of the printed products in tissue engineering. The present microgel baths often exhibit large sizes and poor dispersions, resulting in a big diameter and irregular morphology of the printed filaments. Therefore, it poses a significant challenge in preparing a microgel bath with a small particle size and uniform morphology.This protocol offers the fabrication of a homogenous and biocompatible cationic cross-linked kappa-carrageenan sub-microgel bath for high-fidelity bioprinting, through an easy-to-operate mechanical grinding strategy, Since kappa-carrageenan sub-microgels exhibit a uniform methodology with a small particle size of 565 nanometer, and a low state of 0.35%enabling precise tissue and organ construction with remarkable fidelity and high resolution.

颗粒状 κ-角叉菜胶凝胶浴是通过将块状水凝胶机械分解成颗粒状凝胶浆液而生成的。最近的研究表明，κ-角叉菜胶颗粒的平均直径约为 642 ± 65 nm，在 1000 rpm 的机械混合15 下具有均匀的形态，明显小于先前文献中报道的微凝胶的尺寸 16,17,18.更长的破碎时间和更高的速度导致更小的微粒尺寸，在?...

Read this Scientific Article Protocol about Embedded Bioprinting of Tissue-like Structures Using κ-Carrageenan Sub-microgel Medium at JoVE.com

本研究介绍了一种新型 κ-角叉菜胶亚微凝胶悬浮浴，显示出显着的可逆干扰-非干扰过渡特性。这些属性有助于在嵌入式 3D 生物打印中构建仿生组织和器官。成功打印具有高分辨率和细胞生长的心脏/食管样组织，展示了高质量的生物打印和组织工程应用。

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic ...

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Research

JoVE Journal

Engineering

Embedded Bioprinting of Tissue-like Structures Using &#954;-Carrageenan Sub-Microgel Medium

589 次观看.  Ningbo University. 基于微凝胶的悬浮浴中的 3D 生物打印为使用软水凝胶制造复杂的 3D 结构提供了一个多功能平台，但它仍然受到低打印分辨率和准确性的限制，阻碍了打印产品在组织工程中的实际应用。目前的微凝胶浴通常表现出大尺寸和较差的分散性，导致打印细丝的直径大和形态不规则。因此，制备小粒径和均匀形态的微凝胶浴带来了重大挑战。该方案通?...

视频: 作者聚焦：用于高分辨率 3D 生物打印的均相 κ-角叉菜胶亚微凝胶浴的开发

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic scaffolds. However, engineering a suitable gel suspension medium that balances precise bioink deposition with cell viability and function presents multiple challenges, particularly in achieving the desired viscoelastic properties. Here, a novel &#954;-carrageenan gel supporting bath is fabricated through an easy-to-operate mechanical grinding process, producing homogeneous sub-microscale particles. These sub-microgels exhibit typical Bingham flow behavior with small yield stress and rapid shear-thinning properties, which facilitate the smooth deposition of bioinks. Moreover, the reversible gel-sol transition and self-healing capabilities of the &#954;-carrageenan microgel network ensure the structural integrity of printed constructs, enabling the creation of complex, multi-layered tissue structures with defined architectural features. Post-printing, the &#954;-carrageenan sub-microgels can be easily removed by a simple phosphate-buffered saline wash. Further bioprinting with cell-laden bioinks demonstrates that cells within the biomimetic constructs have a high viability of 92% and quickly extend pseudopodia, as well as maintain robust proliferation, indicating the potential of this bioprinting strategy for tissue and organ fabrication. In summary, this novel &#954;-carrageenan sub-microgel medium emerges as a promising avenue for embedded bioprinting of exceptional quality, bearing profound implications for the in vitro development of engineered tissues and organs.

This study introduces a novel &#954;-carrageenan sub-microgel suspension bath, displaying remarkable reversible jamming-unjamming transition properties. These attributes contribute to the construction of biomimetic tissues and organs in embedded 3D bioprinting. The successful printing of heart/esophageal-like tissues with high resolution and cell growth demonstrates high-quality bioprinting and tissue engineering applications.

科研

工程学

Preparation of the &#954;-Carrageenan Sub-Microgel Suspension Bath for Embedded 3D Printing

Begin by adding 1.75 grams of Kappa Carrageenan powder to 500 milliliters of PBS in a 1000 milliliter glass bottle to prepare a Kappa Carrageenan suspension bath. Then introduce a 70 millimeter magnetic stir bar into the glass bottle. Tighten the glass bottle cap and then loosen it by half a turn.Heat the glass bottle in a 70 degree Celsius water bath. Then turn on the magnetic stir at 300 RPM. Place the bottle and stir until the polymer is completely dissolved.Using a 0.22 micrometer filter, filter the completely dissolved Kappa Carrageenan solution into the sterilized glass bottle. Store the Kappa Carrageenan solution at four degrees Celsius to induce a cation cross-linked gelation for 12 hours. Then using a 60 millimeter magnetic stir, mechanically grind the Kappa Carrageenan hydrogels until they successfully transform into a liquid state.

用于嵌入式 3D 打印的 κ-角叉菜胶亚微凝胶悬液的制备

Printing Biomimetic Hydrogel Scaffolds Using a Custom-Designed Micro-Extrusion Bioprinter

To begin, switch on the micro-extrusion pump controller. Select the corresponding volume syringe and set the extrusion rate at 0.08 milliliters per minute. Then divide the hydrogel volume by the extrusion rate and coordinate it with the desired printing time to calculate the duration of extrusion.Next, fit the syringe with a needle featuring an inner diameter of 210 micrometers into the syringe slot of the bioprinter. Adjust the syringe controller to ensure that the plunger of the syringe is in close contact with the screw. Then place three milliliters of the prepared kappa-carrageenan suspension bath into a 35 millimeter cell culture dish.Based on the code settings, position the dish on the platform and confirm that the printing head is one millimeter above the bottom of the dish. After that, insert the SD card containing the code into the 3D bioprinter. Activate the code file, start the bioprinter, and click the Start button on the controller.Then expose the printed construct to 405 nanometers of blue light for one minute to initiate photo-crosslinking. Using a one milliliter pipette, remove the kappa-carrageenan gel, followed by the addition of three milliliters of PBS for washing and subsequent removal.

使用定制设计的微挤压生物打印机打印仿生水凝胶支架

Embedded 3D Bioprinting of Esophageal Muscle-Layer Analogs for Tissue Engineering

To begin, take the cultured esophageal smooth muscle cells once they attain 80%co fluency. Aspirate the medium and wash the endothelial smooth muscle cells with five milliliters of PBS. Then add two milliliters of 0.2%Trypsin-EDTA to the flask and incubate for two minutes to digest the cells.Add two milliliters of DMEM to terminate the digestion process and transfer the cell suspension to a 15 milliliter centrifuge tube. Centrifuge the tube at 675G for three minutes. Carefully aspirate the supernatant, and resuspend the cell pellet in the composite gelatin methacrylate and silk fibroin methacryloyl bioinks.After bioprinting and washing the 3D esophageal muscle layer with PBS, carefully aspirate the PBS and replace it with three milliliters of DMEM, supplemented with 10%FBS, and 1%penicillin or streptomycin.