The work presented here is supported by the Singapore National Research Foundation under CREATE program (NRF-Technion): The Regenerative Medicine Initiative in Cardiac Restoration Therapy Research Program and by the Public Sector Funding (PSF) 2012 from the Science and Engineering Research Council (SERC) under the Agency for Science, Technology and Research (A*STAR).

注:此协议描述了使用背压辅助机器人分配系统（ 图1A），为表面刻蚀（ 图1B）和基于挤出的生物印刷器（ 图1C）10。 1.对聚苯乙烯表面改性<ol><li>使用1毫米聚苯乙烯板聚苯乙烯以来组织培养板倾向于向上弓为中心，既破坏蚀刻和印刷的高度一致性。 注:由于聚苯乙烯片不被修改为细胞粘附，进行等离子体处理。 </li><li>氧等离子体处理。 <ol><li>首先，选择2巴的上连接到等离子体机器氧气瓶的调节器压力。然后打开并输入以下条件到机器的控制面板等离子机（分别为电力，燃气流量和时间）为150W，氧气30 SSCM，10分钟。 </li><li>将聚苯乙烯substratË进入等离子体室，封门，按下控制面板上的启动按钮。浸泡在胎牛血清氧等离子体处理的聚苯乙烯基板和洗涤三次1×磷酸盐缓冲盐水（PBS）之前在37℃孵育2小时。 </li></ol></li></ol> 2.编程模式打印<ol><li>使用该程序首先蚀刻表面用触控笔或手术刀。 <ol><li>当使用手写笔，插入直径为1.5mm针纺织品（从非常小心内部）插入到分配注射器（或5或10毫升）的嘴，直到它被卡住并固定。如果使用手术刀，选择一个圆处理解剖刀，以便它可以被紧固到打印臂的夹紧机构。 注:手写笔蚀刻图案弯曲比手术刀更好。 </li><li>当第一次尝试创建一个bioprinted安排，素描在坐标纸上所需的图案与编号轴产生XY坐标。然后我NPUT蚀刻/ bioprinted图案的坐标到电子表格软件（ 图2）。 注意:"所需的图案"可以是多种形状，如直链的，S形，或圆形的。的选择取决于所需的实验模型，如平行的线性线的MSC的分化为心肌如这里证明。散点图功能允许提出情节模式的可视化。 </li><li>打开打印/分发软件。选择"程序&gt;添加程序"，其次是"编辑&gt;添加点"来设置程序。导出x和y坐标从电子表格中获得的值成打印/分配使用软件"复制和粘贴"的功能。 </li><li>校准每次运行前，机器人的"z"的高度，以触针/打印喷嘴放置在表面上。 <ol><li>在打印/分发软件，选择"机器人"选项，点击"更改模式＆＃34;并启用"教学模式"选项。一旦选择，软件使机器人的"点动"功能。 </li><li>慢跑，首先通过选择菜单栏下面的命令机器人初始化到默认位置; "机器人&gt;梅卡初始化"，然后选择"机器人&gt;点动"。输入中以精确地放置触笔上的图案的原点所需要的"X和Y槽"的数值（单位mm）。 </li><li>接着，在"Z槽"输入的数值（毫米）放置铁笔或打印喷嘴中与表面接触，但不挠曲或缩进表面。这点被指定为"Z"的初始值。 注:各槽的深度可以很容易地使用该系统的Z高度变化。 40，80和170微米槽证明切成1毫米厚的聚苯乙烯片材（ 图4和表1）的表面上。 </li></ol></li><li>选择在P每个坐标点RINT指令， 即 "行配给的开始"来定义的第一点和打印起始，"行传递"指定的中间点和"行配给结束"的信号给机器人终止印数。 </li><li>通过从顶部菜单栏中选择后续指令控制程序的机器人:"机器人&gt;发送C＆T的数据"。 </li><li>启动蚀刻/印数，通过改变机器人的"运行"模式。通过选择这样做从顶部菜单栏中的"机器人&gt;更改模式&gt;切换运行模式"。 </li><li>通过按下绿色"启动按钮"机器人饮水机控制台上启动打印程序。 </li></ol></li></ol> 3.制备和印刷的明胶生物油墨含有细胞的<ol><li>溶解2％的明胶在最小必需培养基阿尔法培养基（αMEM）（补充有10％FBS和2％抗生素/ antimycotIC）在60℃下2小时以制备生物油墨溶液。 </li><li>预培养的红色荧光蛋白表达间充质干细胞（RFP-MSCS）至70％汇合使用αMEM/ 10％FBS10厘米组织培养皿。通过在37℃下除去培养基和涂料用1X胰蛋白酶-EDTA溶液中5分钟，释放附着的细胞成悬浮液。 </li><li>沉淀通过离心将细胞在1000×g离心5分钟，去除上清。重悬在0.5ml 1×PBS中的细胞沉淀，并使用血细胞计数器计数细胞密度。 </li><li>允许生物油墨冷却至室温后，轻轻以足够的量混合到悬浮液中的RFP-MSC的在生物油墨以实现5×10 6个细胞毫升-1的终浓度。 </li><li>倒入细胞生物油墨轴承与密封喷嘴的打印注射器。冷却加载注射器至4℃，以达到一印刷的粘度。 </li><li>放置装注射器上的自动化全自动点胶系统，并将空气压力线。取出注射头密封和连接打印喷头。 </li><li>挤出细胞化bioinks成使用0.05MPa的背压细线，以5mm /（锥形建议在圆柱形）从经由27克针/喷嘴10毫升注射器秒写入速度到聚苯乙烯膜表面，之后预编程的沉积在步骤2.1中所述图案至细胞化生物油墨放置到预刻蚀槽。 </li><li>在室温下孵育1小时后，使用倒置荧光显微镜观察之前加入10 mL生长培养基（补充有10％FBS和抗生素），孵育细胞24小时（以使细胞以感测和反应的蚀刻特征）在放大10倍。 </li></ol>

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The authors declare they have no competing financial interest.

这个过程的关键步骤是干细胞作为过程必须允许细胞沉降到该特征，打印无生物油墨扩频/出血，递送细胞无剪切应力的细胞死亡，而不是触发分化朝不希望的谱系的实际生物印刷递送。 如果预期细胞对准未能发生，则该生物油墨粘度应该评估其适合印刷。重要的是，该生物油墨允许细胞沉降到该图案化的聚合物表面上。该生物油墨聚合物的浓度，可以减少或它​​的温度升高（最多37℃），以降低粘度和促进细胞沉淀。但是，这些削减可能会导致生物油墨扩散/出血后打印。在这里，我们建议使用2％的明胶溶解在1X PBS（V W /）。如果细胞浓度印后出现降低，然后通过印刷引起的剪切应力可能已受到损害的印刷单元7的可行性。检查在生物油墨细胞之前和使用活力测定法，诸如刃天青荧光7的激活打印后。此外，如果bioprinted干细胞接触该表面但不对齐，则有可能在打印过程中的剪切应力通过触发不期望的分化途径减少了其多能。交的打印干性可通过荧光激活细胞分选（FACS）分析用干细胞标记物进行评估，如CD 29 10,19,20。如果它发现该剪切应力具有对细胞有害作用，然后可以通过添加或具有剪切稀化的，如藻更换生物油墨聚合物可以降低生物油墨印刷粘度，普朗尼克F127，透明质酸，结冷胶，明胶丙烯酸甲酯，聚氧化乙烯19，21-25。 最后，可以优化其他方面包括所述打印背压，生物油墨细胞密度，和喷嘴直径。背压需要优化的生物油墨的粘度，并且可以被改变以获得一个离散的不间断的跟踪。高细胞浓度可能增加生物油墨粘度和堵塞打印头。然而，该挤压过程中允许比其它生物印刷方法-26,27-相对更高的细胞密度的打印。打印喷嘴计的选择应加以考虑，因为小口径喷嘴可能获得更精细的痕迹，但会容易堵塞。锥形喷嘴已发现更好地运作，因为相同的流速，可以实现作为一个圆柱形喷嘴，但在因此提高细胞生存力28的下剪切应力。 背压辅助沉积法，也被称为挤出生物印刷，具有比其他细胞印刷方法如喷墨和激光辅助方法少的分辨率由于最低可编程步长和EMERG的扩展ING生物油墨。然而，背压挤出设备最容易修改列入一个蚀刻手写笔。使用光刻法，随后通过PDMS模塑生产槽如微图案化的其它方法具有比所述机器人蚀刻显著更多的步骤，并且该方法是在修改现有的图案或产生新的12,15,29-31少的通用性。 此方法提供了细胞的相互作用，其中的位置，取向，和一种或多种细胞类型的配置是重要的体外研究的工具。几种细胞类型，包括间质干细胞，成纤维细胞和平滑肌细胞已知在表面的槽1,10,14,18对齐。蚀刻图案，这里提出，可以进行修改并再版非常迅速和轻松。这是感兴趣的，与槽表面图案化已在细胞黏附，形态，迁移的研究已经使用和干细胞的分化。此外，我T公司也被应用到神经肌肉骨骼组织工程18，32,33。由于槽做在细胞分化或表型调节对数种细胞类型的作用，这种方法可用于创建沟槽，以有助于分化并且使得屏幕能够进行离散行沉积的细胞。目前，我们正在评估此方法，以促进细胞用于生产心肌细胞片的各向异性孔结构，如蜂窝取向为心脏组织12-14,34的功能是重要的。 这里展示的方法使用一种可溶性生物油墨，最终将消失。然而，如果需要更持久的水凝胶覆盖，然后微生物转谷氨酰胺酶的包含可交联并稳定蛋白质的水凝胶，例如基于明胶那些没有在细胞上9，35。Bioinks还可以利用甲基丙烯酸酯化的聚合物为UV引发crosslinkin不利影响在细胞相容性的方式36克细胞轴承水凝胶。然而，根据我们的经验，我们发现，相对于聚时所赋予的水凝胶和聚合物表面（不易脱层），和更细胞相容性环境之间的改进的相互作用的转谷氨酰胺酶交联（乙二醇）二丙烯酸酯（PEGDA）水凝胶37。

单元布局的蓄意构图，可允许在体内细胞组织1模仿文化的形成。事实上，研究多种细胞类型之间的相互作用可以通过组织他们的空间位置2,3的协助。大多数图形系统依赖于表面改性方法，为推动或阻止随后的被动细胞沉积的细胞粘附。提供生物印刷在细胞分布1空间和时间的控制。除了这些功能，生物印刷已被描述为用于产生几何形状复杂的支架4在技术上简单，快速和成本有效的方法。它利用计算机设计软件，并允许引入细胞进入制造工序4。 生物印刷系统已根据自己的基础工作原理激光被分类，喷墨型或挤压型4。挤压生物印刷已被描述为是最有前途的，因为它允许一个现实的时间帧4-6内临床相关尺寸的组织构建物的制造。它是由一个细胞轴承水凝胶生物油墨的机械或背压辅挤出进行。在这里提出的方法中，被采用的背压。如所提到的，将细胞在细胞相容性生物油墨递送。这样的生物油墨应该支持细胞的递送，而不产生有害的剪切应力，并具有足够的粘度以保持在印刷迹的完整性，而不会破裂或扩散（称为"墨渗色"），7-10。 已知细胞与它们的贴壁表面的相互作用来影响细胞的行为。表面形貌可以控制细胞形状，方向11，甚至表型。尤其是，沟槽和通道的制造中已被证明诱导在多个细胞类型拉伸的，细长的形态。这种形态的通过已发现影响多能和多潜能细胞的表型。例如，当在槽对齐，间质干细胞（MSC）表现出对心肌12,13和血管平滑肌细胞分化的证据采用收缩表型在合成10,14-17。 可在聚合物表面通过许多方法来产生对准通道或沟槽的细胞，例如，深反应离子蚀刻，电子束光刻，激光直接印刷，飞秒激光，光刻和等离子干蚀刻18。这些方法通常耗时，需要复杂的设备，并且可以在所产生的图案的形状进行限制。此外，他们不与同步生物印刷图案，不允许立即细胞化。一个自动化的协调控制的运动分配系统可以遵循复杂的图案为溶液的沉积。在这里，我们展示了微型受控的运动是如何被利用来创造细胞定向通道。削尖触笔或手术刀被连接到所述打印头代替挤出注射器中，然后将设备可以蚀刻绘图软件的指导下，在聚合物表面上。该方法在图案设计提供了多功能性，并适用于在生物工程常用如聚苯乙烯，聚四氟乙烯，和聚己内酯的聚合物材料。作为后续步骤的蚀刻，细胞可以直接bioprinted到划伤凹槽。这里使用的明胶生物油墨能够既保持跟踪，并允许沉积的细胞来检测蚀刻特征。 bioprinted的刻蚀槽间充质干细胞，表现在不同的线条沿它们伸长。

<table border="0" cellpadding="0" cellspacing="0" width="448">
	<tbody>
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			<td height="21" style="height:21px;width:151px;">Equipment</td>
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			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:151px;">Robotic Dispensing System</td>
			<td style="width:131px;">Janome</td>
			<td style="width:104px;">2300N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Plasma Machine</td>
			<td style="width:131px;">Femto Science</td>
			<td style="width:104px;">Covance</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">USB Microscope</td>
			<td style="width:131px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Optical Microscope</td>
			<td style="width:131px;">Olympus</td>
			<td style="width:104px;">IX71</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Software</td>
			<td style="width:131px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Spreadsheet</td>
			<td style="width:131px;">Excel</td>
			<td style="width:104px;">Excel</td>
			<td></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:151px;">Printing Co-ordinate Software</td>
			<td style="width:131px;">Janome</td>
			<td style="width:104px;">JR C-Points</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:151px;">Imaging Software</td>
			<td style="width:131px;">National Institutes of Health (NIH)</td>
			<td style="width:104px;">ImageJ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Equipment</td>
			<td style="width:131px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Stylus (Blade)</td>
			<td style="width:131px;">OLFA</td>
			<td style="width:104px;">AK-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">5ml printing syringe</td>
			<td style="width:131px;">San-ei Tech</td>
			<td style="width:104px;">SH10LL-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">30G printing needle</td>
			<td style="width:131px;">San-ei Tech</td>
			<td style="width:104px;">SH30-0.25-B</td>
			<td></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:151px;">1mm polystyrene sheets</td>
			<td style="width:131px;"></td>
			<td style="width:104px;">Purchased locally</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Fetal bovine serum</td>
			<td style="width:131px;">Invitrogen&#160;</td>
			<td style="width:104px;">10270-098</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:151px;">Phosphate buffered saline</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:151px;">Gelatin from porcine skin, Gel strength 300, Type A</td>
			<td style="width:131px;">Sigma Aldrich</td>
			<td style="width:104px;">9000-70-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">&#945;MEM</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:104px;">41061-029</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Antibiotc antimycotic</td>
			<td style="width:131px;">Sigma Aldrich</td>
			<td style="width:104px;">A5955-100ML</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:151px;">Red Fluorescent Protein Mesenchymal Stem Cells (RFP-MSCs)</td>
			<td style="width:131px;">Cyagen Biosciences Incorporation</td>
			<td style="width:104px;">RASMX-01201</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated robotic dispensing technique for surface guidance and bioprinting of cells

这个手稿描述引进细胞指导功能接着用自动机器人分配系统对这些特征的直接递送细胞在水凝胶生物油墨。被选定的特定生物油墨，因为它使细胞对沉积物和感知功能。分配系统bioprints使用背压辅助打印头中的水凝胶bioinks活细胞。然而，通过用锋利笔或手术刀更换打印头，分配系统也可以采用以创建经过表面刻蚀地形线索。触针运动可以在X，Y的10微米的步骤和Z方向进行编程。图案化的凹槽能够定向间质干细胞，影响它们通过在对准的细长的形态与凹槽&#39;方向。图案形成可以在直线，同心圆，和正弦波使用绘图软件来设计。在随后的过程中，成纤维爆炸和间充质干细胞悬浮于2％的明胶生物油墨，用于在背压从动挤压印刷头生物印刷。然后将细胞轴承生物油墨使用用于蚀刻相同的编程坐标打印。所述bioprinted细胞能够感知和通过沿刻蚀槽的方向上其细长方向所证明反应的蚀刻特征。

代表结果表明，该反压辅助机器人分配系统可以作为一个基于挤压的生物印刷器，用于执行两个表面蚀刻和生物油墨印刷（ 图1的A）。它可用于刻蚀槽的产生到聚合物表面上，并且随后直接打印的小区轴承生物油墨的特征（ 图1 B和C）。 既蚀刻和印刷由编程坐标确定（ 图2）输入到打印/分配软件，它使线形的印刷，弯曲，并根据需要为应用程序（ 图3的AC）径向图案。 Z轴设置允许蚀刻凹槽深度的控制（ 图4和表1）。 更换后与水凝胶打印头触笔，即协调蚀刻编程可重复使用，以使机器人分配系统直接输送单元轴承生物油墨与下列相同的设计（ 图3 D和E）的刻蚀槽。如可在图5中A和B观察到的，干细胞中，通过生物油墨内生物印刷最终沉淀到表面接种，并感测和沿着分立线路中蚀刻的方向伸长。在培养基中接种无特征的方向生物印刷对准的细胞，但是，它们所涉及的整个表面（ 图5℃），这证明生物油墨限制了细胞的印刷迹线。如果没有蚀刻的特征，在文化传媒种子细胞没有显示的取向或对齐（ 图5 D）。 <img alt="图1" src="/files/ftp_upload/54604/54604fig1.jpg" /> Figu重1:该自动机器人分配系统 （A）的装置，是与包含一个锋利笔（固定在注射器）中央到蚀刻同心环到聚苯乙烯幻灯片（B）的所述打印头的定制。然后打印头可以转换到挤出细胞轴承明胶生物油墨，用背压辅助挤出，并沉积在先前蚀刻图案（C）。这个数字已经从Bhuthalingam 等进行修改。 2015年10。 <img alt="图2" src="/files/ftp_upload/54604/54604fig2.jpg" /> 图2: 绘图的xy坐标中的电子表格软件这里显示的屏幕抓取表明，绘制XY坐标创建的正弦波图案如由曲线图的情节。这些坐标使用复制/粘贴都输入到绘图软件功能。 <img alt="图3" src="/files/ftp_upload/54604/54604fig3.jpg" /> 图3:刻纹图案通用编程到绘图软件的坐标能够蚀刻线性（A）中，正弦（B）和同心圆（C）。蚀刻成聚苯乙烯槽可以在随后的步骤（D）和（E）可以直接印刷到与水凝胶生物油墨。这个数字已经从Bhuthalingam 等。2015年10修改。 <img alt="图4" src="/files/ftp_upload/54604/54604fig4.jpg" /> 图 4: 蚀刻槽深蚀刻槽的深度可通过分配系统的z轴控制进行调节。图像（A）至（C）表示俯视图和（D）与（E）表示剖面图。机器人被编程到表面蚀刻到40的深度（（A）和（D）），90（（B）和（E））和180微米（（C）和（F））。编程深度和实际蚀刻深度的接近度显示在表1中 。这个数字已从Bhuthalingam 等人 2015年10修改。 <img alt="图5" src="/files/ftp_upload/54604/54604fig5.jpg" /> 图5:生物油墨输送间充质干细胞在生物油墨印刷到蚀刻图案的RFP-MSC可相比接种于细胞感测功能可以通过细长形态和对准槽（A）和（B）中所证明。功能，而生物油墨（ 即在培养基）（C &lt;/强&gt;）和一个非图案化的表面上的（D）。这个数字已经从Bhuthalingam 等。2015年10修改。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 编程的深度（微米） </td><td> 腐蚀沟槽的深度（微米） </td></tr><tr><td> 40 </td><td> 35±7 </td></tr><tr><td> 90 </td><td> 81±6 </td></tr><tr><td> 180 </td><td> 175±3 </td></tr></tbody></table> 表1:的编程相对于聚苯乙烯实际蚀刻深度比较该图已经从Bhuthalingam 等人 2015年10修改。

Watch this Scientific Journal Video about Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells at JoVE.com

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

自动点胶机器人技术在细胞表面和指导生物印刷

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

automated-robotic-dispensing-technique-for-surface-guidance

Nanyang Technological University

Research

JoVE Journal

Bioengineering

6.3K 次观看.  Nanyang Technological University. This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

视频: Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

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 ...

Robotic Mirror Therapy System for Functional Recovery of Hemiplegic Arms

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is conducted by eliciting an illusion through use of a mirror as if the hemiplegic arm is moving in real-time while moving the healthy arm. It can facilitate brain neuroplasticity through activation of the sensorimotor cortex. However, conventional mirror therapy has a critical limitation in that the hemiplegic arm is not actually moving. Thus, we developed a real-time 2-axis mirror robot system as a simple add-on module for conventional mirror therapy using a closed feedback mechanism, which enables real-time movement of the hemiplegic arm. We used 3 Attitude and Heading Reference System sensors, 2 brushless DC motors for elbow and wrist joints, and exoskeletal frames. In a feasibility study on 6 healthy subjects, robotic mirror therapy was safe and feasible. We further selected tasks useful for activities of daily living training through feedback from rehabilitation doctors. A chronic stroke patient showed improvement in the Fugl-Meyer assessment scale and elbow flexor spasticity after a 2-week application of the mirror robot system. Robotic mirror therapy may enhance proprioceptive input to the sensory cortex, which is considered to be important in neuroplasticity and functional recovery of hemiplegic arms. The mirror robot system presented herein can be easily developed and utilized effectively to advance occupational therapy.

We developed a real-time mirror robot system for functional recovery of hemiplegic arms using automatic control technology, conducted a clinical study on healthy subjects, and determined tasks through feedback from rehabilitation doctors. This simple mirror robot can be applied effectively to occupational therapy in stroke patients with a hemiplegic arm.

机器人镜治疗系统偏瘫武器功能恢复

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

微流控生物工程血管化组织和细胞器解体

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

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 ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

高通量自动 Microbioreactor 系统在 CHO 细胞模型 IgG1 生产中的应用

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

使用机器人微注射操作脑片中单神经干细胞和神经元

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...

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 ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.