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。 有设计过程，影响所产生的构建体的仿生函数的关键方面。以控制生物材料和基板的温度的能力对于水凝胶的胶凝机制和它们的我的维护必要械性能，因此影响细胞分布，增殖和水凝胶内的分化。器官由许多细胞类型的，所以多个分配器可用于制备异构的，组织样结构是至关重要的。外部结构的计算机辅助设计允许生产定制组织替代不同的伤口或组织。这是患者特异性的替代品的发展至关重要。内部架构也是同样重要的，因为它通过将合适的细胞彼此紧密接触，并允许他们在体内形成样细胞-细胞连接的影响结构内的细胞-细胞之间的关系。细胞的精确位置决定了细胞如何沟通和网络互相形成血管网络，并模仿它们的生物活性的天然组织。三维生物印刷提供了生物油墨内均匀分散的细胞，以及对空间p优良的精密lacement细胞。生成的支架也具有高的地方的细胞密度，这对于细胞分化和细胞外基质的配制剂是必不可少的。 这里介绍的是一款3D机器人生物印刷器的可靠和一致省去均匀滴用仿生水凝胶混合单个细胞或细胞。类似bioprinters已经使用的预融合技术。按照差动粘附假说，当放置在模具或类似装置的单个细胞，将保险丝和组织的基础上在细胞面传热的浓度和粘附分子的分布。这些设备创建预稠合杆或其他几何形状都被装入分配器，并放置在接近其他预稠合或预制的棒，然后熔断成一个更大的几何十八。端的几何形状是由什么可以从这些预先制作实体构成限制。这里实施的生物印刷器包括一个独特的温度受控的环境，其中，细胞和细胞水凝胶的混合物不被融合前的必要性的限制。在这些条件下，所述生物印刷是不完全依赖于差分粘附假说。列入水凝胶材料可以帮助引导细胞分布和允许细胞融合，或没有，这取决于所需性质的特定的实验。仿生水凝胶为细胞封装的选择还对细胞表型产生深远的影响。材料是已知对细胞附着，以及细胞大小和形态学的效果。流变学特性，如粘度，水凝胶决定它们对蜂窝microenvironmen影响。原生藻酸盐是惰性的，并且不容易与参加表型的对照细胞进行通信。然而，使用被通过肽偶联和氧化化学改性藻酸盐，将所得构建体显示控制降解性和增加的细胞附件，迁移和proliferatio。改变生物材料的物理化学性质会影响组织的发展。 使用的流体分配三维生物印刷，直接写入机器由印刷结构的分辨度的限制，水凝胶材料，初始细胞死亡后打印的可用性，以及血管化的仿生的能力。的生物印刷的一个重要特点是它的分辨率。各印刷方法是通过最小的实现细节下的技术极限尺寸定义。有印刷结构的下限大小和规模达到之间的动态关系:对微小的细节分辨率越高，较小的最大结构。该生物印刷器能够以非常具体的和有组织的方式沉积量小至230 NL，给它一个更高的分辨率比同类机器。水凝胶已被常用于生物印刷由于其亲水性，生物相容性，结构相似的细胞外基质，和易于改性中的。水凝胶的高含水量提高他们的组织相容性，但大大降低了其机械强度和。目前缺乏优化水凝胶的过程中bioadditive制造挤压相应的力学性能流体输送。因此，存在对于开发水凝胶是免疫惰性的，具有可以使用流体输送成功地挤出细胞相容性凝胶化的机制，并且还产生具有机械的最佳范围内的单元格载货基质的大量需求。在印刷过程之前，所述细胞载货水凝胶生物油墨必须存储在注射器的时间量，损害细胞的。在印刷过程中，剪切应力在挤出过程中诱导的对细胞也可能是有害的。的生物印刷器能够产生高度可行的（&gt; 90％）的结构，因此克服i的问题nitial细胞死亡。血管化起着传输，支持或保留的bioprinted仿生功能至关重要的作用。氧扩散是米;因此在较大的bioprinted构造缺氧是一个。传统技术不能生产构建体具有嵌入脉管，大大限制了可生产支架的大小。的生物印刷器的细胞活力评估表明在超过8天印刷构建显著细胞增殖。因此，该技术证明了其生成的支架，允许细胞生长，通信和网络的形成，血管化的要求的能力。 的生物印刷器提供了使用的各种材料，以快速沉积在特定模式的细胞载货水凝胶的能力。虽然这种技术产生异质结构具有可调性，它是不能同时沉积和活性混合。对于一些生物材料，这种沉积会见HOD将增强凝胶机制，缩短时间，支架生产中。增加了一个多注射器分配器可能允许更广泛的生物材料为biofabrication技术。细胞活性的以上的时间将提供关于水凝胶的特性，细胞网络的形成，并且所述构建体的血管的更多信息较长时间内bioprinted构建体调查。 所描述的生物印刷器的沉积方法可以进一步包括机器人定位和驱动三个分配器以沉积多个生物材料上的预先沉积的材料顶部以预定的图案。这一步可以通过使用升序模式直到三维器官或组织产生重复。因此，棕榈打印机是适用于可靠地分配小区载货水凝胶以创建一个三维结构，其能够保持脉管和高细胞生存力的，并可以在组织工程应用中使用。

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><tbody><tr><td>Positioning Robot (JR2000 XYZ)</td><td>Janome&amp;nbsp;</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:&amp;nbsp;</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:&amp;nbsp;</td><td>Fishman Corporation</td><td>122051</td><td></td></tr><tr><td>22 G Dispenser Tips</td><td>Fishman Corporation</td><td>Z520122&amp;nbsp;</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&#039;s Modified Eagle&#039;s Medium</td><td>Gibco Life Technologies</td><td>11965-092</td><td>Warm in 37 &amp;deg;C water before use.</td></tr><tr><td>Trypsin/EDTA</td><td>Lonza</td><td>CC-5012</td><td>Warm in 37 &amp;deg;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 &amp;deg;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 &amp;deg;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&amp;nbsp;</td><td>10 G</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline, +Calcium, +Magnesium</td><td>Life Technologies</td><td>14040133</td><td>Warm in 37 &amp;deg;C water before use.</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline, -Calcium, -Magnesium</td><td>Life Technologies</td><td>14190144</td><td>Warm in 37 &amp;deg;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 &amp;deg;C until use</td></tr><tr><td>(4&amp;rsquo;, 6-Diamidino-2-Phenylindole, Dihydrochloride) (DAPI) Stain</td><td>Life Technologies</td><td>R37606</td><td>Store at -20 &amp;deg;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的，是适当的生物油墨的直接表示。 RGD肽结合在一段时间通过促进细胞扩散过长提高细胞活力。荧光显微镜来定量细胞活力在印刷过程之后的结构。海藻酸钠生物油墨用精矿的15％和5％的氧化通货膨胀有天0生存能力98％，96％4天，和95％8天（ 图5）。这些结果表明的直写生物印刷器的沉积技术挤出细胞轻轻足以产生期间和打印过程后保持存活的构建体（图1，2）。高细胞活力表示5％的氧化和15％浓度的藻酸盐生物油墨是一个合适的载体用于细胞沉积和提供用于细胞存活的适当环境。在每个区域相似的细胞计数显示在藻酸盐生物油墨均匀细胞分布，打印分辨率的一个基本方面。 大多数组织中有复杂的组合和细胞外基质成分，每个特定的生物和机械影响的梯度。生物材料应该是仿生的天然环境，促进细胞功能。藻酸盐脚手架的高孔隙率允许细胞进行通信和网络彼此，并且还可以促进在支架和其周围环境之间的营养物质和代谢物的通量。细胞粘附到细胞外基质是组织形成前细胞增殖和细胞外基质分子的组织进入功能性组织发生这种情况的一个初步阶段。细胞的增殖起着伤口愈合和组织生长至关重要的作用，并且因此分析bioprinted构建体用于组织工程应用时的一个重要因素。含RGD缀合藻增强细胞附着在印刷构建体，从而提高细胞扩散和增殖。细胞在印刷支架的增殖进行定量计数上天三个独立的区域0和8（ 图6）。整体细胞增殖被发现后8天培养为219.674％。这些结果表明该支架有足够的生物相容性中使用的SA合成的细胞外基质的传递细胞修复损坏或无法正常工作的组织。 为了分析的RGD多肽偶联上藻酸盐生物油墨的成功，使用细胞载货，RGD缀合的15％浓度，5％的氧化藻酸盐生物油墨和细胞载货，非共轭15％浓度，5％的氧化进行了比较实验海藻生物油墨。 DAPI染色的细胞核和鬼笔环肽染色的肌动蛋白被用来分析的单元上天在印刷构建扩频各样品8.图像（每个样品至少三个随机图象）利用使用30 Z堆叠参数共焦显微镜系统服用光学片通过300深度（图7）。与RGD缀合藻酸盐的样品中的细胞扩散所示证明在藻酸盐的肽的成功掺入。细胞迁移是组织发展的重要一步;因此，藻酸盐RGD肽的结合提高了李kelihood 体内使用这种生物油墨的应用。 <img alt="图1" src="/files/ftp_upload/53156/53156fig1.jpg" /> 图1.棕榈打印机。可编程逻辑控制器协调所有的打印机功能（A）的行动。气密密封室（B）用经过滤的摄入量（C）和排气（C）的保持经调节的内部正压力，以减少污染的机会。安装在腔室天花板双紫外灯（D）可以被编程以安全的间隔进行操作。一个JANOME 2300N XYZ机器人（E）的编程和集成的电脑（我）来控制。三个分配器控制器（G）的被编程以调节分配器枪（F）的可用的输出被加载到所述机器人的Z轴臂（H）的计算机控制下。机器人样品架的温度（J）是由一个水浴控制器（K），组4和40℃之间。双数码相机（L）是用T<html> O监控打印机的活动和样品的形成。一台摄像机被安装在机器人的Z轴臂，并提供了上膛的枪的分配端的放大图像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53156/53156fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53156/53156fig2.jpg" /> 图2.工具组，光学传感器和生物印刷器位移激光。从前面A.卸载工具组视图。从前面B.加载工具组视图。C.光学传感器测量所述分配头端在三维空间。D.距离激光测量印刷表面的Z坐标。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53156/53156fig2large.jpg" target="_blank">请这里查看该图的放大版本。</a> ove_content"FO:保持together.within页="总是"&gt; <img alt="图3" src="/files/ftp_upload/53156/53156fig3.jpg" /> 计算机辅助设计绘图软件的用于设计bioprinted构建体的外部体系结构图3的视觉PathBuilder软件。形象。该方案提供了生成增量间隔液滴和复杂的几何形状的能力。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53156/53156fig3large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53156/53156fig4.jpg" /> 图4. JR-C点软件，打印机兼容设计软件的屏幕截图。这个程序允许控制沉积法的用户（ 即单滴沉积或连续途径沉积），沉积速度快，注射器前端和印刷苏之间的距离<html> bstrate表面，在规定的时间为每滴，滴和（ 见表1）的三维位置的沉积。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53156/53156fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53156/53156fig5.jpg" /> hADSC的在使用共焦显微镜系统（30光学片上的深度Z堆栈参数）后0（A）的取bioprinted构建体和8 彩绘hADSCs后打印。细胞生存力/细胞毒性的图5.荧光图像的荧光图像（ B）天。该hADSC的被使用哺乳动物细胞生存力/细胞毒性测定标记后打印。活细胞被染成绿色，和死细胞被染成红色。3156fig5large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图6" src="/files/ftp_upload/53156/53156fig6.jpg" /> 图6. Bioprinted构造的量化存活率。活的和死的细胞数，使用生存力/细胞毒性测定定量。活/死细胞计数为0天示于（A）中，并在（B）中的计数第8天。计算每个区域的天0和8活细胞的数量显示在（C）和用于量化细胞增殖。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53156/53156fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/53156/53156fig7.jpg" /> 细胞拉登，N图7.比较使用共焦显微镜系统（Z堆叠参数上共轭和RGD-共轭藻酸盐。的bioprinted hADSC在非共轭（A）的荧光图像，和在RGD缀合（B）的15％浓度的5％的氧化藻酸盐生物油墨取30光片上的深度）。该hADSC的沾满毒伞素和DAPI染色分析细胞在每一个结构的蔓延。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53156/53156fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>表命令</td><td></td></tr><tr><td></td><td></td></tr><tr><td>命令</td><td>机器人响应</td></tr><tr><td> PTP点</td><td>机械臂移动到指定的posiiton在X，Y，Z空间</td></tr><tr><td> Find_Base_Z </td><td>采用SICK激光测量牛逼他印制基板的表面上;注射器尖端和基板表面之间的距离被手动设置。 </td></tr><tr><td>工作ADJ。号（工作调整数） </td><td>命令机器人使用SICK激光（用于测定基材表面），1枪，枪2，或3枪。 </td></tr><tr><td> Get_1 </td><td>命令机器人OT检索和加载枪1 </td></tr><tr><td> Find_Tip1_YZ </td><td>机器人发现枪1中的Y和Z方向的前端</td></tr><tr><td> Find_Tip1_X </td><td>机器人发现枪1的在X方向上的前端</td></tr><tr><td>点分注</td><td>机器人将分配生物油墨的一个液滴中所确定的X，Y，Z位置</td></tr><tr><td>托盘号（Pallte号） </td><td>采用了手动编码设计，印刷， 例如，一个数组。 </td></tr><tr><td>胶时间</td><td>是分配给每个单独的液滴的沉积的时间。&lt;/ TD&gt; </tr><tr><td> Store_1 </td><td>命令机器人喷枪1返回toolnest，并返回到原点:（0,0,0）。 </td></tr></tbody></table> 表1.可编程计算机软件命令。这表列出了可编程计算机软件命令，将其用于控制所述机器人臂和优化印刷适性参数。

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

Nanyang Technological University

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

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

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

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

人体软骨组织制作采用三维喷墨打印技术

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

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 ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

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 ...

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

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

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

使用3D生物印刷创建心脏组织展示球体的机械整合

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

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 ...

使用 κ-角叉菜胶亚微凝胶培养基推进 3D 生物打印

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

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.

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

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.

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

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

工程学

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

Bioprintable 藻酸盐/明胶水凝胶 3D体外模型诱导细胞球形形成

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create tissues by adding layer upon layer of printable biomaterials, termed bioinks, and cells in an orderly and automatic manner, which allows for creating highly intricate structures that traditional tissue engineering techniques cannot achieve. Thus, 3D bioprinting is an appealing in vitro approach to mimic the native vasculature complex structure, ranging from millimetric vessels to microvascular networks. 
Advances in 3D bioprinting in granular hydrogels enabled the high-resolution extrusion of low-viscosity extracellular matrix-based bioinks. This work presents a combined 3D bioprinting and sacrificial mold-based 3D printing approach for fabricating engineered vascularized tissue flaps. 3D bioprinting of endothelial and support cells using recombinant collagen-methacrylate bioink within a gelatin support bath is utilized for the fabrication of a self-assembled capillary network. This printed microvasculature is assembled around a mesoscale vessel-like porous scaffold, fabricated using a sacrificial 3D printed mold, and is seeded with endothelial cells. 
This assembly induces the endothelium of the mesoscale vessel to anastomose with the surrounding capillary network, establishing a hierarchical vascular network within an engineered tissue flap. The engineered flap is then directly implanted by surgical anastomosis to a rat femoral artery using a cuff technique. The described methods can be expanded for the fabrication of various vascularized tissue flaps for use in reconstruction surgery and vascularization studies.

Engineered flaps require an incorporated functional vascular network. In this protocol, we present a method of fabricating a 3D printed tissue flap containing a hierarchical vascular network and its direct microsurgical anastomoses to rat femoral artery.

使用3D打印技术制造工程血管瓣

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create ...

Bioprinted蜂窝构造的可行性使用三分配器笛卡尔打印机

摘要

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人体软骨组织制作采用三维喷墨打印技术

生物印刷细胞化构造使用组织特异性水凝胶生物油墨

自动点胶机器人技术在细胞表面和指导生物印刷

使用3D生物印刷创建心脏组织展示球体的机械整合

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

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

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

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

Bioprintable 藻酸盐/明胶水凝胶 3D体外模型诱导细胞球形形成

使用3D打印技术制造工程血管瓣