This work was supported by NIH-NIDCR IRO1DE019355 (MJ Yost, PI), and NSF-EPSCoR (EPS-0903795).

1.确定物中的胶原蛋白批次的百分比<ol><li>提取继先前公布的程序13的胶原蛋白。解冻的最低20ml的胶原蛋白。确定以操纵在形成的水凝胶，胶原浓度在批处理胶原固体的初始百分比。 <ol><li>切三块铝箔（约6×6厘米）中，并通过用25毫升烧杯的底部形状每一个作为锅。记录每个锅的重量。 </li><li>胶原的少量添加到每个平移和记录铝锅和胶原的总重量。添加0.5 - 0.8克胶原每个铝盘。 注意:在此步骤之后，有三个铝箔盘和每一个应当有胶原的量小。确保记录每个（总共3）空铝盘的重量和锅的加成胶原后的重量。 </li><li>使用F计算胶原的重量在每一个锅ollowing公式: 胶原蛋白的重量=潘和胶原蛋白的重量 - 潘重</li><li>放置持有在100℃下在烘箱中胶原24小时三个铝盘中。 </li><li> 24小时后，记录每个铝盘的重量和脱水的胶原蛋白。 </li><li>计算使用下面的等式脱水胶原的重量: 脱水的胶原蛋白的重量=平移和脱水的胶原蛋白的重量 - 潘重</li><li>计算的固体的百分比为三个样品，使用以下公式确定胶原固体浓度: <img alt="公式（2）" src="/files/ftp_upload/53578/53578eq2.jpg" /></li><li>计算使用胶原固体为三个样本的百分比批次的平均胶原固体含量。 注意:将要使用的胶原是留下水合胶原蛋白。脱水的胶原蛋白没有将被使用。 </li><li>确定胶原固体（我的百分比后该批nitial胶原浓度），继续使用剩余的水合胶原。使用经过校准的pH计的胶原批次的pH调节至3。 <ol><li>加入少量（每次2-5微升）12 N盐酸（HCl）的。保持冰在任何时候。不直接添加盐酸以胶原 - 酸添加到该管的一侧。加酸后，用锅铲酸推到胶原蛋白，迅速搅拌混合。 </li></ol></li><li>一旦它的pH值达到3，让胶原坐O / N在4℃。 </li></ol></li></ol> 2. BSA橡胶的制备<ol><li>制备按照下面所列的步骤的牛血清白蛋白（BSA）溶液。 <ol><li>制备2倍磷酸盐缓冲盐水液（PBS）中。添加两个PBS片剂至100毫升的水，使0.02M的PBS溶液。 </li><li>结合BSA的2倍的PBS来创建使用下列顺序的30％BSA溶液。 <ol><li>例如，为了使30％的BSA用30ml 2×PBS中，使用12.9克的BSA。添加2倍的PBS（例如，10ml）中的1/3到有搅拌棒的烧瓶中。添加BSA（如，6.45克BSA）在烧瓶1/2和使用刮刀，润湿干溶质。 </li><li>重复加入PBS中，然后BSA的这个过程，直到所有的溶质和溶剂在烧瓶中。用锅铲湿所有的溶质。它看起来像一些液体和团块的混合物。让它坐大约30分钟。 </li><li>然后，打开一个小周期的搅拌器，并确保周围有搅拌棒无团块。它应该采取90分钟得到解决方案的所有或者也可以在4℃下搅拌O / N。溶质全部溶解后，放置在20毫升注射器的溶液，并用0.20微米的注射器过滤器盖上。按下柱塞通过过滤器排出液体，并收集在一个新的管中的灭菌溶液。保存在4℃。 </li></ol></li><li>准备3％戊二醛soluti通过稀释无菌过滤2X PBS 25％戊二醛溶液。例如，10毫升3％戊二醛溶液，使用2个ml等于25％的戊二醛和8毫升2×PBS中。保存在4℃。 </li></ol></li></ol> 3.治疗模具 注:本文中所描述的原型采用的是定制的不锈钢Ÿ防霉片。模具包含一个流入和分别为4和3mm两个流出通道。首先，清洁模具，喷洒与不饱和猪油和消毒他们。按照以下描述的步骤制备的模具。 <ol><li>使用超声波仪，在35千赫的频率干净的不锈钢模具。放置模具在超声波仪，用水和冰淹没它们。保持模具冷在任何时候都超声波仪运行时。运行超声波仪90分钟2​​期。 <ol><li>每一个时期后，用针，以确保没有在鲁尔无重大锁定不锈钢或bRASS连接器。用肥皂和水清洗模具双方的整个表面。验证是否有在渠道上没有障碍。 </li></ol></li><li>放置模具，螺丝，和鲁尔锁定连接​​器在高压釜中袋和高压灭菌它。 </li><li>填补重视与市售猪油（混合脂肪酸脱模剂）的空气喷雾器一半的瓶子。更换一个普通盖瓶帽。将其放置在高压釜中袋和高压灭菌它。 注意:猪油用于促进该材料，就可以注射以后反应（BSA橡胶）的释放。不要把喷雾器瓶盖在autoclave-它会损坏内部密封。 </li><li>热身猪油45秒，直到它的明确和液体微波炉。螺杆空气喷涂机盖猪油瓶。盖连接的喷雾器。附加喷雾器在实验台上的气源。打开空气阀，并打开喷雾器的喷嘴，直到它开始润湿表面的纸巾。 </li><li>喷猪油垂直于模具表面，直到表面被完全覆盖。每一块被喷后，将它们放置在培养皿和密封。放置模具在4℃下2小时。 </li><li>继续进行由表面向UV光暴露30分钟以消毒该模具。将它们放回在4°C，直到他们准备好要反应注射。 </li></ol> 4. BSA橡胶反应注射 注:所有的材料和解决方案应保持低温，直到准备使用，以防止BSA橡胶过早凝固在接下来的步骤。 <ol><li>准备分配器交付BSA橡胶以下步骤模具。 <ol><li>消毒所有混合组分（二O形环，注射器帽，双注射器，混合尖端，和4:1分配器）通过暴露于UV光在聚合酶链反应（PCR）罩30分钟。 注意:PCR罩被使用，因为Ť他的过程涉及固定剂。这些化学物质不能在细胞/组织培养橱中使用，由于曝光和毒性的细胞的风险。包含UV光的任何其它罩将是合适的。 </li><li>放置在溶液保持器的尖端帽。 </li><li> BSA的比为1:在一个4进行混合和注射戊二醛。 30％BSA加入双注射器腔，将提供的解决方案的最高金额（这将需要大约4毫升填写）。确保留有足够的空间，以O形圈放置，以防止相邻的腔室的溢流和污染。 </li><li>加入3％戊二醛溶液到另一个房间（这将需要约1毫升填写）。请务必留出足够的空间放置O形环，以防止相邻室四溢和污染。 </li><li>放置在分配器中的双注射器。垂直倾斜组件使得注射器帽是在顶部。更换混合头帽。 </li><li>钪REW两个不锈钢模具拼在一起。 </li><li>将装配高压釜袋里面。 </li><li>以除去任何空气在分配器，将其保持在直立位置，并迅速挤压手柄一次，以释放与戊二醛混合波塞少量。然后迅速装上不锈钢Ÿ模具的鲁尔锁连接器连接到注射器尖端。 </li><li>持用左手和右侧的BSA的戊二醛混合物分配器不锈钢ý模具。通过按排出到高压釜袋的侧面，以确保内部的空隙填充有溶液交替覆盖各流出通道的左侧和右侧排气。然后，水平放置的模具和再次注入。 </li><li>从分离饮水机和地点该模具在25毫米的培养皿。 </li><li>放置封口膜周围的培养皿，以防止橡胶的脱水。 </li><li>放置在4℃冰箱O / N模具。 </li></ol></li></ol>注意:胶原应在所有时间的过程中被保持在冰上。 <ol><li>修改使用胶原固体的百分比胶原浓度。 <ol><li>通过调整用冷水初始浓度胶原蛋白使10毫升1.75％的胶原蛋白。 </li><li>使用校准pH计，调节用12N盐酸将pH至3。不直接添加盐酸到胶原的酸添加到该管的一侧。加酸后，用锅铲酸推入的胶原蛋白，迅速搅拌混合。 </li><li>权衡4克胶原在一个单独的锥形管中。 </li><li>离心胶原在4℃和9,343×g离心20-30分钟以除去空气。 </li><li>紫外线消毒细胞培养罩30分钟，并添加14微升粘连到4毫升胶原。这将导致10微克/微升的最终层粘连蛋白的浓度。注:层粘连蛋白将提供关于Structural完整性，附着力，并促进各种细胞应答。 </li><li>打开PCR紫外灯上20-30分钟用消毒罩之前。 </li><li>放置路厄锁盖，附加到20ml的注射器，在乙醇中2小时。然后，使其干燥，并放置在UV光。 </li><li>伽玛辐照胶原蛋白8.6分钟达到1200 cGy的。 注意:时间将取决于铯源的衰减。调整以达到相同的剂量的时间。 </li></ol></li></ol>对BSA橡胶6.Casting胶原蛋白<ol><li>以聚合胶原，使用8:1:1的比例（胶原:HEPES:MEM）中。下面的过程是基于一个初始4克酸化胶原（来自步骤5.1.8）的。 <ol><li>使在水中的0.2N的HEPES溶液，并通过加入1-5 1M氢氧化氢氧化钠（NaOH）溶液少量（1-5微升）将pH调节至9。使用校准pH计，监测每次加入后的溶液的pH值。保存在4℃或科EP冰上。 </li><li>打开组织培养罩的UV光20-30分钟进行消毒罩。 </li><li>蒸压钳，铲，手术刀和消毒。 </li><li>拌1.5的0.2N的HEPES（pH为9）和1.5的10倍的MEM毫升利用组织培养罩毫升。请一定要保持它在冰上旋涡其5秒后才能使用。存放在4℃或保持在冰上。 </li><li>消毒的PCR罩，开启UV光30分钟。 </li><li>放置在-20℃的12孔平板和一个20毫升注射器10分钟，或直至准备继续。注意:将所有材料冷待用。温度的增加导致过早胶原原纤维。 </li><li>喷雾所有管和孔板那将是在用70％乙醇罩，并让他们干灭菌。置于冰上20毫升注射器冷却以备后用。 </li><li>在PCR罩，打开不锈钢模具释放BSA橡胶和使用手术刀，切开BSA R的排气通道ubber模具。 </li><li>下的PCR罩中，打开无菌胶原管和加入1毫升的HEPES-MEM溶液（确保提取的HEPES-MEM溶液，这是很好的混合，并且没有固体沉淀之前）。 </li><li>使用无菌刮刀，调匀胶原蛋白和缓冲溶液。 </li><li>关闭并涡旋它迅速，以确保充分混合的水凝胶。 </li><li>转移到冷20毫升注射器。 </li><li>用一只手握住的BSA橡胶阱的内部，并使用其他分配一半胶原凝胶溶液到井的底部。 </li><li>使用无菌镊子，确保橡胶流入和流出的两端接触的侧面很好。 </li><li>倒入橡胶的顶部胶原溶液直至完全覆盖。 </li><li>确保BSA橡胶胶原内暂停，不存在任何气泡，尤其是靠近橡胶的端部。 </li><li>将机盖和包装封口膜周围的阱的周边。 </li><li>把在培养箱中1小时，在37℃。保持对PCR紫外光。 </li></ol></li><li>胶原聚合后，紫外线交联通过以下过程中的水凝胶。 注意:胶原的交联将使用其中可以控制能量的量的UV交联剂装置来完成。 <ol><li>打开UV交联剂和使用的能源与环境μJ630000 /厘米2照射空室。 </li><li>从培养箱中取出凝胶。 </li><li>喷雾手用乙醇，而且，在室内，迅速取下盖越好。 </li><li>关闭室和紫外线通过选择能量设置和照射630000μJ/ cm 2的交联水凝胶。 </li><li>交联循环后，关掉紫外线灯的PCR罩</li><li>喷雾手用乙醇和打开密室，迅速将盖放回孔板。移动孔板到PCR罩。 </li><li>使用无菌刮刀，轻轻拧松并从井中取出凝胶。翻转引擎盖下的凝胶以交联水凝胶的底部。重复步骤6.2.3和6.2.4。 </li></ol></li></ol> 7. BSA橡胶酶切<ol><li>为了具有中空胶原支架，除去BSA橡胶中的方式，不影响嵌在水凝胶的尺寸。该过程描述如下。 <ol><li>打开紫外光20-30分钟来灭菌组织培养罩。 </li><li>使0.25％的胰蛋白酶溶液的pH值7.8。例如，15毫升水，在50毫升锥形管中添加0.0376克胰蛋白酶。通过加入1M NaCl的少量（2-5微升）调节pH值至7.8。放置在20毫升注射器的溶液，并用0.20微米的注射器过滤器盖上。按下柱塞通过过滤器排出液体，并收集在一个新的管中的无菌溶液。 </li><li>放水浴，将温度设定为30C。 </li><li> 30分钟后，关掉紫外线灯。喷洒在将在罩中使用的所有管和材料乙醇。 </li><li>在不同的锥形管引擎盖下发生转移的胶原凝胶。 </li><li>周围添加3-5毫升（刚好足以覆盖凝胶）的0.25％胰蛋白酶溶液，pH值为7.8至每个管。 </li><li>密封用封口膜和涡轻管约1分钟。 </li><li>发生在30℃的水浴中15-24小时。而在水浴，轻轻频繁涡凝胶直到BSA的橡胶已被消化或从水凝胶中移除。 注意:为了确定是否波塞橡胶已被去除，或者将橡胶漂浮在胰蛋白酶溶液或有破旧件。必须有水凝胶内没有视觉暗区。 </li></ol></li><li>为了确保所有的BSA的橡胶和胰蛋白酶已经从水凝胶取出，如下所述冲洗。 <ol><li>打开PCR呼ð紫外线20-30分钟。 </li><li>准备Mosconas解决方案。在水结合氯化钾（KCl，28.6毫摩尔），（ 碳酸氢钠 ，11.9毫摩尔），葡萄糖（9.4毫摩尔）和（的NaH 2 PO 4，0.08毫摩尔）。调节pH值至7.4，用1M的NaOH或12M的HCl溶液。放置在20毫升注射器的溶液和使用0.20微米的注射器过滤器进行消毒的溶液中。 </li><li>喷雾用乙醇一切之前将它们放置在引擎盖上，并允许乙醇干燥。 </li><li>打开管并转移5-10无菌Mosconas溶液中到一个新的50ml锥形管中。 </li><li>转移胶原水凝胶包含酶溶液的锥形管中。确保该水凝胶完全覆盖该溶液中。留在冰箱摇床管在4℃下30分钟。 </li><li>吸出Mosconas解决方案，并重复步骤7.2.5两次。 </li><li>保存在4℃。 </li></ol></li></ol>

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作者什么都没有透露。

生物制造是在生物学和工程学原理合并产生模仿自然组织复杂的材料高度多学科领域。为了达到这个目的，有必要开发使用从体内组织收集到的信息，并将其转换成一个在体外的支架技术。以这种方式，平台可改造成非常类似于体内组织的建筑，功能，和机械性能。最佳的支架材料应具有一定的性质，例如是生物相容的，模仿的感兴趣的组织的机械性能，能够受控降解的，能够支持细胞活力，并能够允许组织重塑2,3。 制造技术的众多已发展到产生可行的，立体的结构。这些技术可分为两大类:常规的ð推进。现有技术包括使用合成的和天然的传统材料，以使多孔结构。一些实例是溶剂浇铸，冷冻干燥，和熔融成型。这些技术的缺点包括在结构内的孔隙率的控制差（孔径和孔互连性）和难度使得支架内的内部通道。先进的技术包括光固化，成型，3D打印，静电和等等1。这些技术有缺点，例如，缺乏长程微通道，材料选择，以提供最佳的机械强度，同时能够通过小直径喷嘴，优化依赖于材料分配的，需要大量的后处理可能是有毒的，并且限制在在体外构建体内部结构的设计。在3D打印的一个主要缺点是足够的细胞生物材料载体的可用性的同时，还具有保持规定的建筑机构12所需的机械性能。这里介绍的技术结合常规和先进的制造技术。两全其美是从计算机辅助制造到创造，或进口，与酶不稳定橡胶的发展所需要的架构的融合而得。不锈钢模具用铣床创建的，但它们的制作方法并不限于这种技术。使用3D打印机（例如，Makerbot），相同的模具从聚乳酸（PLA）制造。负模具用的CAD程序，它提供了创建特征的一种简单而可靠的传输到任何物质固体模具设计建筑指令研磨。这种技术是显著在于它不仅允许外部组织组合物的控制，而且还高度复杂的内部结构。工作这里提出主要集中于BSA的橡胶，其可以混合均匀，在合理的时间量被容易消化的表征，是在其结构中的改变抗性，并且是能够模仿分钟的功能，同时在铸造过程中保持其稳定性。这种技术的局限性进行了测试和牺牲材料的分辨率可维持的尺寸小到300微米的直径。 然而 ，图7表明该信道是由脱模剂的薄层围绕。使用显微镜，这种额外的地区是明确区分，并可以拆下来有需要的尺寸。这种生物制造技术允许的各种各样的，范围从宏观到微观尺度内部结构的复制。 血清白蛋白是在循环系统中最丰富的蛋白质。由于蛋白质是聚电解质，溶解度通过静电相互作用来确定15-17，它已经显示，在低盐浓度，有一种盐析蛋白质中促进其溶解性18上的效果。戊二醛是导致在白蛋白的性质的改变的交联剂。 在图1中看到的颜色变化归因于醛亚胺键19-21的形成。戊二醛与BSA通过赖氨酸的氨基主要反应形成的分子间共价键14。数据表明，盐的增加而提高了橡胶的弹性（从1×PBS中2倍PBS）中。较高浓度的戊二醛生产更严厉的凝胶是先前设置相比，低浓度的。然而，它们更难以分配，均匀混合，并且它们形成脆的凝胶。交付的BSA橡胶适量到模具中，组件的每一个部分必须保持冷，因为更高的温度下加速的戊二醛与BSA反应。理想的橡胶（30％BSA在2倍PBS或1x PBS 3％戊二醛），表现为能够承受负荷而不会永久变形粘弹性材料。处理和周围的BSA的橡胶结构铸造材料时，这变得非常重要。 胰蛋白酶是一种丝氨酸蛋白酶水解的蛋白质。胰蛋白酶是具有较高的切割特异性一种广泛使用的酶。它主要裂解肽链在氨基酸赖氨酸的羧基侧和精氨酸22。 BSA是在低浓度下易溶于水，而胰蛋白酶容易消化的BSA的橡胶留下胶原完好和相对不变。该材料不会与细胞的任何接触。该构建体测试了游离戊二醛的存在和没有这种固定剂的痕迹。这显示了BSA的橡胶作为生物制造牺牲材料的功效。在这个协议中，胶原被用作支架材料，但任何加时赛可以用她的材料，是抗胰蛋白酶的消化。 今后的工作将集中于由播种内皮和成纤维细胞的支架重构的水凝胶中的血管的组件。面临的挑战之一是在整个该模拟天然三维分布通道的内部创建的细胞的均匀分布。为了解决这个问题，一个策略是正在开发，允许密封或通道的堵塞和细胞悬浮液的注射。测试的材料是普朗尼克F127，它是热可逆凝胶，液体在4℃和固体在30℃以上23,24。普朗尼克高浓度的成功创造了必要的时间印章。细胞悬液是该支架的通道内之后，直到细胞粘附在三维结构的所有侧面的实体被旋转为一个特定的时间量。内部通道将有保持足够的媒体细胞的存活。普卢兰尼克保持其凝胶形式，并且在水性环境中容易溶解。一旦细胞附着，水凝胶将与媒体被淹没，并且可以在静止的或流动的条件下，进行培养取决于研究的目的。这一方法将进一步评估，而且将成为了遵循本出版物。本文描述的生物制造技术目前正在用于开发人类肾动脉的支架复制品。同样的方法可以与其他类型的组织，如心脏来完成，该技术的适用性扩展到了广泛的临床应用。 在这里开发的生物制造技术在体外支架，能够快速，可靠地概括内在几何特征的一代向前迈进了一步。天然材料，如胶原蛋白，被选中是因为它提供了最佳的化学和物理线索细胞合成材料上。这些呐王兴仁材料可用于治疗的研究，开发，畸形和疾病组织的体外模型 ，以及用于替换受损组织。

在组织工程领域，制作组织支架的能力是至关重要的。合适的组织支架具有三维结构，是由生物相容的材料制成，并在体内组织架构模拟物，以促进细胞和组织的生长和重建。该支架必须允许营养物质的运输和清除废物1-4。一种在生产这些支架的主要障碍是概括特定几何特征成生物相容的材料的能力。几个生物制造技术已经报道，以控制这些支架的几何特征，实施例是静电5-8，溶剂浇铸9，立体光刻10，和3D印刷11，等等。这些技术达不到在提供相对容易的可控内部和外部的建筑特色转移，是昂贵的，由它们的分辨率和印刷性能（限<em&gt;例如，喷嘴计，材料限制），或需要制造后技术，这需要的时间，以产生可行的支架12长的时间。 在许多商业制造系统，内部空隙，通道和特征的创建是用砂或其他合适的可移动或牺牲材料来实现。的金属或塑料部件围绕砂模形成，并且一旦它被固化时，砂被去除。在大致相同的方式，下一代生物材料的所需要的生物沙等效。因此，波塞橡胶被开发作为用于生物沙的替代物。 BSA的橡胶是由具有戊二醛交联的牛血清白蛋白的一个新制定的材料。最终的目标是具体的建筑特色重建成可生物降解的胶原支架。维持二维保真度与原组织的模具牺牲biorubber的特性进行说明。 <p cl屁股="jove_content"&gt; BSA和戊二醛浓度的几种组合进行使用各种溶剂进行测试。该物质经BSA和戊二醛之间的反应产生。 BSA的橡胶可以是注入组织模具的复杂的几何形状的反应。交联的BSA的胰蛋白酶不稳定，并通过在温和的pH和温度条件下的酶容易地消化。相反，完整的I型胶原蛋白是胰蛋白酶消化非常有抗性。这些特征被资本以选择性地移除的BSA橡胶留下胶原后面。目前的工作包括确定获得不稳定的模具，可以到生物相容支架提供特定的建筑特色所需要的理想的参数。进行了评价，该具体特征包括混合性，酶消化，承载，和被注入阴模的反应能力。 30％BSA和3％戊二醛的组合满足这些要求。这个协议提供了necess元指导创建这些三维支架。所述原型包括表示与一个流入和直径分别为4-和3毫米，二流出通道的支链结构的胶原蛋白支架的。该技术具有模仿感兴趣的组织的宏观和微观环境的潜力。这种技术提供了一个可行的技术，以在以高的保真度，其可被调谐以模仿体内组织的弹性和感兴趣的组织的其他特性的相对容易且及时物质递送的特定几何启发可生物降解的材料。

<table><tbody><tr><td>Collagen type I</td><td></td><td></td><td>Collagen extracted from calf hide</td></tr><tr><td>Hydrocloric Acid (HCl)</td><td>Sigma-Aldrich</td><td>7647-01-0</td><td></td></tr><tr><td>Phosphate Buffer Solution (PBS Tablets)</td><td>MP Biomedical</td><td>U5378</td><td>1 tablet per 100 ml makes 1x PBS</td></tr><tr><td>Albumium from bovine serum (BSA)</td><td>Sigma-Aldrich</td><td>A9647</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma -Aldrich</td><td>G5882</td><td>Toxic</td></tr><tr><td>Lard</td><td>Fields</td><td>3090</td><td></td></tr><tr><td>Stainless Steel Molds</td><td></td><td></td><td>Milled using Microlution Machine</td></tr><tr><td>Air Brush Kit</td><td>Central Pneumatic</td><td>47791</td><td></td></tr><tr><td>Mixing Tip for double syringe</td><td>Medmix</td><td>ML2.5-16-LLM</td><td>Mixer, DN2,5X16, 4:1 brown, med</td></tr><tr><td>Small O ring for double syringe</td><td>Medmix</td><td>PPB-X05-04-02SM</td><td>Piston B, 5 ml, 4:1, PE natural</td></tr><tr><td>Double Syringe cap&amp;nbsp;</td><td>Medmix</td><td>VLX002-SM</td><td>Cap, 4:1/10:1, PE brown, med</td></tr><tr><td>Big O ring for double syringe</td><td>Medmix</td><td>PPA-X05-04-02SM</td><td>Piston A, 5 ml, 4:1</td></tr><tr><td>Double Syringe</td><td>Medmix</td><td>SDL X05-04-50M</td><td>Double syringe, 5 ml, 4:1</td></tr><tr><td>Double Syringe Dispenser</td><td>Medmix</td><td>DL05-0400M</td><td>Dispenser, 5 ml, 4:1, med , plain</td></tr><tr><td>Laminim</td><td></td><td></td><td>3.6 mg/ml - extracted USC lab</td></tr><tr><td>20 ml Syringe Luer Lock Tip</td><td>BD</td><td>302830</td><td></td></tr><tr><td>Luer Lock Caps</td><td>Fisher</td><td>JGTCBLLX</td><td></td></tr><tr><td>HEPES</td><td>Sigma -Aldrich</td><td>H4034</td><td></td></tr><tr><td>Gibco Minimum Essential Media 10x (MEM)</td><td>Life Technologies</td><td>1143-030</td><td></td></tr><tr><td>Trypsin</td><td>Life Technologies</td><td>27250-018</td><td></td></tr><tr><td>UV Crosslinker&amp;nbsp;</td><td>Spectroline UV</td><td>XLE1000</td><td></td></tr><tr><td>Sodium Cloride (NaCl)</td><td>Fisher</td><td>S271-10</td><td>To prepare Mosconas</td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma -Aldrich</td><td>P5405-250</td><td>To prepare Mosconas</td></tr><tr><td>Sodium Bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Fisher</td><td>BP328-500</td><td>To prepare Mosconas</td></tr><tr><td>Glucose</td><td>Sigma -Aldrich</td><td>G-8270</td><td>To prepare Mosconas</td></tr><tr><td>Sodium Phosphate didasic (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S-7907</td><td>To prepare Mosconas</td></tr><tr><td>Sterile Filter for syringes</td><td>Corning</td><td>431224</td><td></td></tr></tbody></table>

three-dimensional biomimetic technology: novel biorubber creates defined micro- and macro-scale architectures in collagen hydrogels

组织支架起到组织再生过程中的关键作用。理想的脚手架必须满足几个条件，如适当有组成，有针对性的模量，以及明确的建筑特色。该概括体内组织的内在结构的生物材料可用于研究疾病，以及便于丢失和畸形软组织的再生是至关重要的。一种新型的生物制造技术的开发，结合​​了先进的成像，三维（3D）打印，和选择性酶活性的国家创造了新一代​​生物材料的研究和临床应用。开发材料，牛血清白蛋白橡胶，是注入一个维护特定几何特征的模具的反应。这种牺牲材料允许建筑特色的充分转移到天然支架材料。所述原型包括一个三维胶原蛋白支架的带4和3mm渠道再版ESENT支架构。本文强调的自然结构的产生使用这种生物制造技术。这个协议利用计算机辅助软件（CAD）来制造固体模具，这将是用BSA橡胶接着橡胶的酶消化注入反应，而使支架材料内的建筑特色。

Watch this Scientific Journal Video about Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels at JoVE.com

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

三维仿生技术:新型Biorubber创建自定义微观和宏观尺度架构的胶原凝胶

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

three-dimensional-biomimetic-technology-novel-biorubber-creates

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

9.1K 次观看.  Medical University of South Carolina. An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

视频: Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

打印温敏反向模具图案创作的双组分水凝胶的三维细胞培养

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

科研

免疫与感染

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

使用胶原基支架的神经母细胞瘤三维 体外 仿生模型

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

癌症研究

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

生物工程

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.

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 ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

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 ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

明胶美沙克里洛醇水凝胶生物油墨3D生物印刷方案

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

三维仿生技术:新型Biorubber创建自定义微观和宏观尺度架构的胶原凝胶

摘要

探索更多视频

此视频中的章节

打印温敏反向模具图案创作的双组分水凝胶的三维细胞培养

使用胶原基支架的神经母细胞瘤三维体外仿生模型

工程三维细胞化胶原凝胶的血管组织再生

Bioprinted蜂窝构造的可行性使用三分配器笛卡尔打印机

3D胶原凝胶和微通道3D相互作用的研究准备

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

明胶美沙克里洛醇水凝胶生物油墨3D生物印刷方案

三维仿生技术:新型Biorubber创建自定义微观和宏观尺度架构的胶原凝胶

摘要

探索更多视频

此视频中的章节

打印温敏反向模具图案创作的双组分水凝胶的三维细胞培养

使用胶原基支架的神经母细胞瘤三维 体外 仿生模型

工程三维细胞化胶原凝胶的血管组织再生

Bioprinted蜂窝构造的可行性使用三分配器笛卡尔打印机

3D胶原凝胶和微通道3D相互作用的研究准备

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

明胶美沙克里洛醇水凝胶生物油墨3D生物印刷方案

使用胶原基支架的神经母细胞瘤三维体外仿生模型