这项工作是由下批准号:EPS-0903795荣获由美国国家科学基金会，美国国立卫生研究院NIDCR R01-DE019355（MJY PI），和格兰特8P20 GM103444（YM PI）政府的支持来支持的。

1.制备明胶含有底物的藻酸盐水凝胶的三维生物印刷<ol><li>准备由Pataky 等 11描述，以避免具有高的内容相关联的存活率下降了钙/明胶底物法以下钙/明胶衬底。钙/明胶底物法如下所示。 <ol><li>结合氯化钙脱水物（1.5重量％），氯化钠（0.9重量％），和猪明胶（2重量％）在蒸馏水和煮沸2分钟，以创建一个100mM的明胶溶液。 </li></ol></li><li>倒入5毫升明胶/钙溶液的入100毫米标准培养皿，漩涡的溶液周围，使均匀涂覆的表面上，并放置在一平的表面在冰箱到凝胶O / N（允许胶凝至少8小时后再使用）。 </li><li>为了增加基板表面的不透明度，加入二氧化钛（0.3％重量）的明胶/ 氯化钙溶液。搅拌10分钟。 AUToclave在液体循环明胶/ TiO 2的溶液中30分钟以消毒它。 <ol><li>加入3毫升明胶/ TiO 2的溶液中，以预先制备的明胶板的表面上。摇动混合物，以确保其均匀地在整个表面上扩散。允许在4℃冰箱O操作凝胶/ N（允许凝胶在使用前至少8小时）。基材必须在3天内使用。 </li></ol></li></ol> 2，海藻酸氧化<ol><li>氧化海藻酸钠生物油墨以下通过下述Bouhadir 等人 30的方法，部分氧化的藻酸盐。 <ol><li>使5％的氧化藻酸盐溶液，溶解1克藻酸钠在100毫升蒸馏水中。添加高碘酸钠的水溶液（0.25摩尔，0.25毫摩尔），氧化剂，以产生5％的氧化溶液中。搅拌19小时，在室温。加入40毫升乙二醇的溶液24小时后，结束意图ction。 </li><li>溶解2.5克氯化在溶液中的钠。添加乙醇过量（2:1的比例），以沉淀氧化藻酸盐。离心液在1000 XG收集沉淀物，并在蒸馏水中再溶解。重复乙醇洗涤。 </li><li>冷冻干燥该氧化藻酸盐丸粒并储存在-20℃，直到准备使用。 </li></ol></li><li>通过测量钠的百分比确定氧化度被终止由乙二醇之前高碘消耗。 <ol><li>准备一个碘化钾溶液（20％w / v的，pH值7.0的磷酸钠缓冲液）和一个thyodene溶液（10％重量/体积，pH 7.0的磷酸钠缓冲液）。混合两种溶液与在RT氧化藻酸盐。 </li><li>逐渐下降的反应，藻酸盐和高碘酸钠溶液到碘化钾和theodyne解决方案的混合物。测量该混合物的吸光度分光光度法在426纳米。当它已达到一个最大，记录的藻酸盐和高碘酸钠溶液为V 1的使用量。 </li><li>该反应是<img alt="式（1）" fo:content-width="0.8in" src="/files/ftp_upload/53156/53156eq1.jpg" width="120" /> 。的未反应的高碘酸钠的量为<img alt="方程式1.5" fo:content-width="1.2in" src="/files/ftp_upload/53156/53156eq1.5.jpg" width="150" /></li><li>减去未反应的高碘酸钠的量从原来的浓度，以确定钠高碘消耗量。使用先前的公式，确定藻酸盐的最终的氧化程度。 </li></ol></li></ol> 3.海藻酸肽共轭<ol><li>共轭配体，带有裸精氨酸-甘氨酸-天门冬氨酸序列（肽）插入预先制备的氧化藻酸盐通过以下由罗利等人 31如下所述，以促进细胞粘附和传播RGD-海藻酸钠缀合方法。 </li><li>使用水性carbodiimide化学与G 4 RGDSPto共轭31。 </li><li>溶解1克5％氧化藻酸盐中的0.1M 2-（N-吗啉代）乙磺酸（MES）缓冲液，pH = 4添加1-乙基 - （二甲基氨基丙基）碳二亚胺（EDC，0.54毫摩尔）和N-羟基琥珀酰亚胺（ NHS，0.27毫摩尔）在2:1的比例，以形成酰胺中间体。 </li><li>经由末端胺加0.28毫摩尔的肽，耦合到藻酸盐聚合物的骨架。在室温下搅拌O / N。 </li><li>停止加入2.5克氯化钠到溶液中的偶合反应。添加乙醇过量（2:1的比例），以沉淀氧化藻酸盐。在4000×g离心离心反应混合物5分钟，以收集沉淀物。吸媒体在细胞培养罩和重新溶解在蒸馏水中沉淀。重复乙醇洗涤。 </li><li>冷冻干燥该沉淀物，直到它被完全干燥（显示为白色粉末状物质），并在-20存储℃的冰箱购买使用。 </li></ol> 4.人体脂肪组织基质细胞（hADSC的）细胞培养<ol><li>培养人体脂肪组织的基质细胞（hADSC的）在75厘米处理过的细胞培养瓶（T75烧瓶），覆盖用15ml低葡萄糖DMEM，用10％胎牛血清和1％青霉素 - 链霉素，1％谷氨酰胺和1％抗霉素。直到它们达到汇合（80-90％），每两天改变介质，在细胞培养罩。 </li><li>一旦汇合，转移T75烧瓶的细胞培养罩和暂停hADSC的使用胰蛋白酶酶消化方法。 <ol><li>在引擎盖，吸所有细胞培养基的脱细胞。冲洗用5ml Dulbecco氏磷酸盐缓冲盐水的钙和镁（DPBS ++）。吸去DPBS ++脱细胞。 </li><li>而在罩，通过混合1 ml胰蛋白酶和4ml DPBS ++使胰蛋白酶和DPBS ++的溶液。每个烧瓶要求5毫升SOLUT的离子，所以进行适当的体积为汇合烧瓶的数目。加入5毫升的胰蛋白酶/ DPBS ++到每个烧瓶中，并把它们在孵化2分钟。 </li><li> 2分钟后，取出烧瓶并轻轻敲击它们的两侧以从塔底松开细胞。在显微镜下看每个烧瓶以确保细胞悬浮。将烧瓶放回细胞培养罩并加入3毫升适当的细胞培养基向每个烧瓶中。这结束了胰蛋白酶反应。 </li><li>从每个烧瓶中转移细胞载货媒体并放入50ml锥形。离心他们在1000×g离心5分钟。细胞应该出现一个白色的小颗粒在锥形底部。转移回细胞培养罩，吸媒体。重悬在2ml细胞培养基的细胞中。 </li><li>算使用显微镜下血球细胞。一旦细胞已被计数，含有〜1.3的Mi​​llio在培养罩，等分的媒体量n个单元，并转移到15毫升锥形。离心机15毫升锥形再次含有细胞5分钟，以1000×克。 </li><li>在培养罩，补种剩余细胞在多个T-75烧瓶，加入〜350000个细胞的浓度向每个烧瓶中。新增15 ml的DMEM培养基中，并再次返回到培养箱直到融合。 </li><li>一旦离心机周期完成，返回的15毫升锥形的以细胞培养物。吸从细胞沉淀中的媒体，并将细胞重新悬浮在水溶液藻酸盐溶液，以每生物油墨的毫升1.3百万个细胞的浓度，常常terteriating溶液所以有细胞的整个生物油墨均匀分布。加载细胞载货解决方案到无菌的打印机兼容的3毫升注射器，并拧上无菌22克的塑料尖。 </li></ol></li></ol> 5.生物印刷器安装<ol><li>打开生物印刷器中，每个分配器计算机以及recirculat荷兰国际集团水浴。 <ol><li>手动设置循环水浴温为凝胶化机理。 </li><li>手动设置打印参数关联饮水机计算机上的每个饮水机。分配体积至230 NL，号码的后踏的设置为0，和分配速率，以10μL-sec。 </li></ol></li><li>打开设计软件和程序用于查看计算机上的U​​SB照相机的显示。 <ol><li>使用该软件，手动输入的坐标为5×5点阵与2.4毫米滴之间的间距。 </li><li>设定打印参数是:前端和基板表面之间的距离= 0.1毫米;高度注射器沉积的解禁= 20毫米;每个沉积时间量= 1秒。 </li><li>保存该程序，并将其发送至机器人。 </li></ol></li><li>放置在4℃的打印机阶段明胶/ TiO 2的含培养皿。关闭并锁上房门。 </li><li>使用PLC进行INItialize紫外光源，和消毒腔室90秒。 </li><li>一旦消毒完成后，打开腔和加载包含hADSC的悬浮在海藻酸钠到喷枪1.关闭注射器并锁定室门。 </li><li>使用PLC来打开风扇系统，等待30秒，平衡内压。 </li><li>在计算机上，运行包含几何途径和印刷参数的程序。 </li><li>在整个印刷过程中，观看计算机上的U​​SB照相机的显示器来确认准确和均匀的印刷。 </li><li>一旦打印完成，允许构建体胶凝40分钟。 </li></ol> 6.细胞活性评估<ol><li>涵盖不会被成像立即打印后在DMEM和存储在孵化器直到成像的时间的结构。 </li><li>量化构建体的生存力，使用荧光基活力/细胞毒性测定染色它们，一个第二图像利用共聚焦显微镜。 <ol><li>继试剂盒说明书，制成含有钙黄绿素上午和溴化乙锭同型二聚体-1染色解决方案。为了使将10ml染色液，加入20微升乙锭同型二聚体-1和5μl的钙黄绿素的上午至10ml无菌，组织培养级的Dulbecco磷酸盐缓冲盐水（+镁，+钙; DPBS ++）。 </li><li>沉浸在染色液的bioprinted构建体在黑暗中15分钟。 </li><li>图片使用共聚焦显微镜系统在天0和8的染色结构每次取bioprinted构建多张照片，使用30倍光学片Z-栈参数超过300微米的深度，人工计数细胞。如果电池出现黄色或绿色指望他们一样活着，如果红色，指望他们像死了一样。 </li></ol></li><li>计算细胞存活率百分比作为活细胞中的细胞构建体的总数除以数;细胞活力=数活细胞（绿色+黄色）/总细胞数（绿+黄+红）×100％。 </li><li>计算的细胞增殖每个样品如通过在第0天的细胞数除以天8的细胞数量;第8天/活细胞细胞增殖=活细胞计数指望一天0×100％。 </li></ol> 7. RGD肽共轭分析<ol><li>为了分析对藻酸盐RGD多肽缀合的成功，比较RGD缀合藻酸盐和非共轭藻酸盐。使用这样做，图像印刷构建体（4&#39;，6-二脒基-2-苯基吲哚，二盐酸盐）（DAPI）和鬼笔环肽染色。 <ol><li>通过稀释5微升甲醇原液用200μl的DPBS ++的使工作溶液的phalloidins。储存在-20℃直到使用。 </li><li>使DAPI染色的以下等式300微米原液:（0.10509克/升）/（350.3克/摩尔）= 3×10 -4 M = 0.0003 M = 0.300毫= 300微米。让日ËDAPI工作液通过稀释原液1:DPBS ++ 100获得3微米的解决方案。储存在-20℃直到使用。 </li></ol></li><li>完全淹没在4％多聚甲醛的样品。孵育1小时，在室温。用DPBS ++洗涤三次，使溶液静置5分钟，每次洗涤。从井转移凝胶样品载玻片，过的过程中翻转凝胶。浸入凝胶中的0.1％的Triton X-100（0.1克/ 100毫升）的DPBS + + 10分钟。用DPBS ++洗涤三次，允许5分钟，每次洗涤。 </li><li>通过浸没在工作溶液弄脏印刷构建体用鬼笔环肽。盖上箔和孵化4小时。取出毒伞素染色，洗三次DPBS ++。第一次洗要快，后者洗涤应该坐下，每次5分钟。 </li><li>通过浸没在DAPI工作溶液染色印刷构建体用DAPI。盖上箔和在室温下孵育30分钟。洗刷三次用DPBS ++，允许每个洗涤坐5分钟。观察和图像的样本共聚焦显微镜系统。 </li></ol>

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The authors have nothing to disclose.

组织工程的主要重点是通过开发能够恢复，维持或提高当地组织函数的生物替代品来弥补器官短缺和移植需求之间的差距。这导致了支架的具有复杂，解剖学上正确的外部几何形状，而在它的内geometr精确控制的直接制造。三维生物印刷是用于使用层 - 层approac从数字模型生成各种尺寸和形状的三维结构的方法。三维仿生构建体的制造起着组织工程的进步至关重要的作用。 有设...

组织工程使用在功能替代品的开发生物和工程的原理来维持，恢复或提高天然组织和。产生按需三维仿生结构将有助于在组织工程中以及在基于细胞的传感器，药物/毒性筛选，组织或肿瘤模型和其他科学和技术进步的能力。三维组织工程构建的组织是制造方法的一个基本组成部分，因为它必须密切模仿细胞外基质的天然组织的高度组织的相互作用。 可生物降解的和成形的三维支架是在产生新的组织构建因为细胞迁移以形成一个二维的细胞层的关键因素，但缺乏在偏爱三维生长的能力。该支架用作小区的临时基础附着和增殖，因此它必须从材料具有可控孔隙率和生物降解性，和足够的机械IntegrIT公司构成。该支架材料不应该是细胞毒性或创建从主机有不良反应。水凝胶已被普遍使用在组织工程技术，并且由于它们的亲水性，水凝胶允许整个所有制结构流体和气体交换。通过结合不同的水凝胶，合成水凝胶的属性均可修改，以满足不同的应用需求。 传统的组织工程方法涉及创建所用细胞接种后fabricatio脱细胞多孔牺牲支架。许多技术已经被采用，例如纤维粘结，溶剂浇铸和熔融模塑，但被证明是用于组织工程应用最低限度地成功。纤维粘合方法允许纤维在特定的形状对齐，但它们仅能够促ducing非常薄的支架。溶剂浇铸方法产生高度多孔结构，然而最大产生膜仅为3毫米THIC。因此，使用这些技术制造的三维结构是不可行的。熔融模制技术生产三维支架被证明是成功的，但需要这样的高温下不能在生产PROCES掺入生物材料。支架接种后加工在其满足组织工程学的要求的能力，产生三维支架与预定义或控制的微观结构和有限。凭借雄厚的脚手架播种技术的另一个主要问题是血管和机械差的不足。 生物印刷已经被通过使用无毒的，可生物降解，​​热可逆的凝胶，以克服常规的缺陷扩展到三维。少数的实体自由成形制造牛逼目前所采用的echniques是激光辅助生物印刷和喷墨印刷。激光辅助生物印刷技术使用一个脉冲激光源，一个目标板，以及一个接收衬底，以产生立体感。然而，这种技术是由于低的吞吐量，低细胞存活率限定，只能生产制造的结构的有限的安排，因为只有光交预聚物可用于形成交联的水凝胶。喷墨印刷是作为其通过沉积皮升墨再现的基板上的数字图像数据的非接触方法。然而，喷墨印刷不产生高分辨率构建体，构建体经历快速蛋白质变性，和许多细胞的沉积期间被裂解。 目前，新的添加剂制造生物印刷方法已经被开发出来。在这些系统中的细胞，蛋白质，生长因子，和仿生水凝胶典型地集成到矩阵脑膜IALS在制造过程中，用计算机控制的执行器来生成三维支架基细胞载货结构紧密模仿原生的微架构同时沉积。细胞载货水凝胶构成了生物油墨，可以是异质的，由多种细胞类型，或同质的。添加剂制造系统存款生物油墨滴逐滴或层 - 层经由一次性注射器和提示到计算机控制的阶段能够移动在x，y和z方向上的。通过计算机软件，印刷支架的结构可以很容易地根据应用的要求进行操作。不像常规技术，三维医疗技术（核磁共振成像，计算机断层扫描）可以被并入到设计中，生成患者特异性构建体。这些方法也允许制备血管替换的可能性，因为构建体产生具有较高升OCAL细胞密度，从而使细胞 - 细胞相互作用和改善植入后surviva的可能性。 帕尔梅托打印机是使用可编程的机器人的制造方法，以生成三维多相组织构建（图1）一个定制三维多分配器系统。它允许使用多种材料中的独特的组合，以产生异质结构。在生物印刷器的初始化是生物印刷中，因为它允许您设置各种参数，以优化bioprinted结构的印刷适性最重要的步骤之一。 的生物印刷器包括间歇式过程由可编程逻辑控制器（PLC），该用户操作通过交互的触摸屏控制面板上（图1中的A）受控启动，运行和关闭序列。为了防止生物污染的生物印刷器被封闭在带正压力有机玻璃逻辑材料（PMMA）室与高效微粒计重（HEPA）-filtered空气循环系统（图1，B，C）。打印机的内部可以使用内置的紫外线光源（图1 中的D）进行消毒。的生物印刷器的主要组成部分是一个完全可编程的定位机器人，可以再现地放置一个分配端具有10微米（图1 的E）的精度。有三个分配器，其能够沉积体积低至使用旋转螺杆230 NL（图1 中的F）。它们是独立可编程使用支配印刷参数为每个分配器（图1，G）的 ​​单独的计算机。旋转螺杆式分配利用一个马达驱动螺杆旋转生物油墨向下注射器和出注射器尖端的移动。这些分配器被安装在一个气动LY控制工具巢（图2A，B）中 ，使机器人以切换分配器安装到程序控制（图1 中的H）下的Z轴机械臂。 XYZ表机器人接收从计算机上运行设计软件打印指示（ 图1，I）。每个程序都包含分配位置，校准程序，和饮水机变化的协议。产生构建体的设计主要是由XYZ坐标，其中每个分配器将沉积材料。的生物印刷器包括两个光学光传感器（图2C），该确定注射器前端的XYZ坐标。这些传感器发送坐标信息的机器人，它利用这些来计算分配端端部的位置。有一个附加的位移激光器（图2D），该凸出点尺寸的633纳米二极管红色激光束30×100微米，测量与ACCURA距离CY 0.1微米。当光束高度集中在机器人确定打印表面的Z方向的距离。该测量中，与前端部Z中的光学光传感器测量，允许精确ż计算坐标用来放置分配器触头相对于印刷表面。分配器尖端横向移动和垂直地通过X轴导向光学光传感器找到Y和Z的中心，并且横向穿过一个Y轴传感器来找到X轴的中心。斧+用+ CZ = d，来确定所述表面是相对于分配头端的位置:印刷面使用以下公式中的xyz空间中的平面映射。打印机阶段（图1，J），持有样品培养皿达80毫米的直径，并且使用循环水浴，以保持设定的温度（图1，K）。阶段温度可以在范围-20内设定并保持在稳定的。有一个USB摄像头安装到机器人的Z-臂，以提供分配尖端的打印过程（图1 中，L）的放大图。有一个第二照相机安装朝向腔室内部提供在印刷过程中（图1，L）上的生物印刷器的完整视图的顶部。 甲计算机辅助设计绘图软件确定沉积图案，并允许用户生成增量间隔开的液滴和复杂的结构（图3）。三维途径可以手动编码到打印机兼容的设计软件或从单独的计算机辅助设计绘图软件（图4， 表1）导入。打印机兼容的软件允许的印刷参数的变化，例如在注射器前端和实名词之间的沉积方法（单个液滴沉积或连续通路沉积），三维的通路的几何结构，沉积速率，距离率打印表面，以沉淀单个下降，并且高度和速度的注射器时间量被抬起的液滴的沉积之间。每个程序都包含XYZ点胶的位置，提示校准程序，和饮水机变化的协议来提供一个无菌的环境，无需操作员干预，在打印过程中。机器人的可编程逻辑控制器（PLC）接收运行设计软件从计算机指令，并控制事件的来自外部控制器的时序（例如，分配器）。为了做到这一点，PLC使用一个循环机制来控制分配器，机器人定位装置，和环境因素。 利用旋转式螺杆，液体配送系统的立体直接写入生物印刷允许更高效，准确，并比以前的方法更简单的细胞沉积的过程。这项研究显示了定制的生物印刷器能够产生行政长官LL-载货水凝胶结构具有较高的细胞活力。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Positioning Robot (JR2000 XYZ)</td>
			<td>Janome&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispensers: SDAV Linear Drive SmartDispensers</td>
			<td>Fishman Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Light Sensors:&#160;</td>
			<td>Keyensce</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Displacement Laser: OD Mini</td>
			<td>SICK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recirculating Water Bath: Polystat</td>
			<td>Cole-Parmer</td>
			<td>EW-12122-02</td>
			<td></td>
		</tr>
		<tr>
			<td>USB Cameras: Dino-Lite Premier 5MP</td>
			<td>AnMo Electrionics/YSC Technologies</td>
			<td>AD7013MT</td>
			<td></td>
		</tr>
		<tr>
			<td>Printer-Compatible Computer Design Software: JR-C Points</td>
			<td>Janome</td>
			<td></td>
			<td>Comes with purchase of Janome Robot</td>
		</tr>
		<tr>
			<td>Computer-Aided Design Drawing Software: Visual PathBuilder</td>
			<td>RatioServ</td>
			<td></td>
			<td>Can be downloaded at: www.ratioserv.com/index.php/downloads</td>
		</tr>
		<tr>
			<td>Printer 3 cc Syringes:&#160;</td>
			<td>Fishman Corporation</td>
			<td>122051</td>
			<td></td>
		</tr>
		<tr>
			<td>22 G Dispenser Tips</td>
			<td>Fishman Corporation</td>
			<td>Z520122&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>10035-04-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>9000-70-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium Dioxide</td>
			<td>Sigma-Aldrich</td>
			<td>13462-67-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Protanal LF 20/40 Alginate (Sodium Alginate)</td>
			<td>FMC BioPolymer</td>
			<td>9005-38-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene Glycol</td>
			<td>Mallinckrodt Baker, Inc</td>
			<td>9300-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Periodate</td>
			<td>Sigma-Aldrich</td>
			<td>7790-28-5</td>
			<td></td>
		</tr>
		<tr>
			<td>hADSC</td>
			<td>Lonza</td>
			<td>PT-5006</td>
			<td>Store in vials in liquid nitrogen until use.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Gibco Life Technologies</td>
			<td>11965-092</td>
			<td>Warm in 37&#176;C water before use.</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Lonza</td>
			<td>CC-5012</td>
			<td>Warm in 37&#176;C water before use.</td>
		</tr>
		<tr>
			<td>Calcein AM</td>
			<td>Gibco Life Technologies</td>
			<td>C3100MP</td>
			<td>Store in the dark at -80&#176;C until use.</td>
		</tr>
		<tr>
			<td>Live/Dead Mammalian Viability Assay Kit</td>
			<td>Invitrogen Life Technologies</td>
			<td>L-3224</td>
			<td>Store in the dark at -80&#176;C until use.</td>
		</tr>
		<tr>
			<td>MES Hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M2933</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Hydroxysuccinimide</td>
			<td>Sigma-Aldrich</td>
			<td>130672</td>
			<td></td>
		</tr>
		<tr>
			<td>1-ethyl-(dimethylaminopropyl) carbodiimide (EDC)</td>
			<td>Sigma-Aldrich</td>
			<td>E1769&#160;</td>
			<td>10 G</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline, +Calcium, +Magnesium</td>
			<td>Life Technologies</td>
			<td>14040133</td>
			<td>Warm in 37&#176;C water before use.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline, -Calcium, -Magnesium</td>
			<td>Life Technologies</td>
			<td>14190144</td>
			<td>Warm in 37&#176;C water before use.</td>
		</tr>
		<tr>
			<td>RGD Peptides</td>
			<td>International Peptides</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 Phalloidin Stain</td>
			<td>Invitrogen Life Technologies</td>
			<td>A22283</td>
			<td>Store at -20&#176;C until use</td>
		</tr>
		<tr>
			<td>(4&#8217;, 6-Diamidino-2-Phenylindole, Dihydrochloride) (DAPI) Stain</td>
			<td>Life Technologies</td>
			<td>R37606</td>
			<td>Store at -20&#176;C until use</td>
		</tr>
	</tbody>
</table>

viability of bioprinted cellular constructs using a three dispenser cartesian printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

结果证实了生物印刷器能够精确地沉积在特定的三维位置细胞载货水凝胶和始终如一地利用计算机辅助软件。这些软件决定了放置每个液滴并控制许多参数的分配（ 图3,4）。的生物印刷器的重复性适当沉积的生物材料是根本其在组织工程应用的成功。 细胞活力，一个成功的技术，生物印刷的要求之一，分析1小时和8天后打印。高细胞活力是用于制造仿生构建必要...

Watch this Scientific Journal Video about Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer at JoVE.com

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Bioprinted蜂窝构造的可行性使用三分配器笛卡尔打印机

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

viability-bioprinted-cellular-constructs-using-three-dispenser

Research

JoVE Journal

Bioengineering

10.0K 次观看.  Medical University of South Carolina. A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

视频: Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

三维人体皮肤重建模型:一种工具来研究正常皮肤恶性黑色素瘤进展

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

科研

生物工程

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

创建瞬态细胞膜毛孔，使用标准的喷墨打印机

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

在心肌结构，功能的体内定量评估，灌注与活力使用心脏微型计算机断层扫描

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

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 ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

分层海藻酸钠构造:一个异质细胞群的共培养平台

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Imaging Cell Viability on Non-transparent Scaffolds &#8212; Using the Example of a Novel Knitted Titanium Implant

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in nucleus pulposus (NP) extrusion leads to intervertebral disc destruction. Currently available surgical treatments reduce the pain but do not restore the mechanical functionality of the spine. In order to preserve mechanical features of the spine, total disc or nucleus replacement thus became a wide interest. However, this arthroplasty era is still in an immature state, since none of the existing products have been clinically evaluated.
This study intends to test the biocompatibility of a novel nucleus implant made of knitted titanium wires. Despite all mechanical advantages, the material has its limits for conventional optical analysis as the resulting implant is non-transparent. Here we present a strategy that describes in vitro visualization, tracking and viability testing of osteochondro-progenitor cells on the scaffold. This protocol can be used to visualize the efficiency of the cleaning protocol as well as to investigate the biocompatibility of these and other non-transparent scaffolds. Furthermore, this protocol can be used to show adherence pattern of cells as well as cell viability and proliferation rates on/in the scaffold. This in vitro biocompatibility testing assay provides a propitious tool to analyze cell-material interaction in non-transparent and opaque scaffolds.

Here we present a fluorophore based imaging technique to detect cell viability on a non-transparent titanium scaffold as well as to detect glimpses of the scaffold impurities. This protocol troubleshoots the drawback of imaging cell-cell or cell-metal interactions on non-transparent scaffolds.

非透明的支架成像细胞活性 - 使用新型针织钛种植体为例

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

用运动调节血清治疗韧带结构:转化组织工程模型

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

多路人工细胞微环境阵列的制备

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Calorespirometry: 一种功能强大、无创的研究细胞能量代谢的方法

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Design of an Open-Source, Low-Cost Bioink and Food Melt Extrusion 3D Printer

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling costs. This increasing popularity is partly driven by falling barriers to entry such as system set-up costs and ease of operation. The following protocol presents the design and construction of an Additive Manufacturing Melt Extrusion (ADDME) 3D printer for the fabrication of custom parts and components. ADDME has been designed with a combination of 3D-printed, laser-cut, and online-sourced components. The protocol is arranged into easy-to-follow sections, with detailed diagrams and parts lists under the headings of framing, y-axis and bed, x-axis, extrusion, electronics, and software. The performance of ADDME is evaluated through extrusion testing and 3D printing of complex objects using viscous cream, chocolate, and Pluronic F-127 (a model for bioinks). The results indicate that ADDME is a capable platform for the fabrication of materials and constructs for use in a wide range of industries. The combination of detailed diagrams and video content facilitates access to low-cost, easy-to-operate equipment for individuals interested in 3D printing of complex objects from a wide range of materials.

The aim of this work is to design and construct a reservoir-based melt extrusion three-dimensional printer made from open-source and low-cost components for applications in the biomedical and food printing industries.

设计开源、低成本生物墨水和食品熔炼挤出3D打印机

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling ...

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