We gratefully acknowledge funding from the Wellcome Trust Affordable Healthcare for India and an EPSRC Landscape Fellowship for Ilida Ortega as well as contributions from The Electrospinning Company Ltd.

道德声明：在本研究中使用的眼睛是根据上公开​​对动物研究的声明中使用： 1，制作PLGA生物降解膜的配备Micropockets <ol><li>环支架采用的microstereolithography和静电纺丝技术31的组合产生。在本质上，该方法可以归纳为2份（ⅰ）创建的PEGDA模板由microstereolit​​hography以及（ii）电纺丝到模板用于再现的基本​​PEGDA结构（在这种情况下，微制造环）。这两个步骤进行详细说明（ 图1和2）。 <ol><li>的PEGDA模板由microstereolit​​hography制造（microSL） <ol><li>使用2层模型制造的环，与所述第一层（L1）是该结构的基极与所述第二层（L2）的呈递6 micropockets马蹄形态学各种尺寸的ES从300-500微米。 PEGDA戒指的制作是最近这组22中描述。 <ol><li>通过任何合适的画图程序绘制1.2厘米黑圈创造的L1。 </li><li>以相同的方式创建的L2但包括8白0.5×0.35毫米马蹄形结构。黑圈结构中散发白色的小马蹄形状。 </li><li>保存L1和L2为JPEG格式。 </li></ol></li><li>在黑暗的玻璃小瓶中，混合聚乙二醇二丙烯酸酯（PEGDA，锰= 250克/摩尔），用1％w / w的樟脑醌，光引发剂，在磁力搅拌器上20分钟。 </li><li>展开microstereolit​​hography使用伸缩式镜头结构建立的激光束，然后投射到它的计算机的可编程数字多镜装置（紫外线功能的DMD入门套件）。注：DMD的反射图像（在这种情况下的环）上的反射镜，通过10cm的焦距管透镜。的图像，然后由银辅酶定向泰德反映到含有光固化聚合物（PEGDA）小瓶。 </li><li>调整并仔细清洗microstereolit​​hography设立的光学系统。 </li><li>把300微升的-PEGDA水混合成一个12孔培养板的孔中。确保将孔预先涂有特氟隆或其他非粘着材料，便于拆卸的结构的固化后。 </li><li>以前安装在PC上，并上传L1到ALP3，基础软件，交换机上的蓝色激光（150毫瓦MBL-Ⅲ473纳米）。照射所述第一层为60秒。注：ALP3，基本是一个USB接口，提供了PC和数字微镜器件之间的联系。 </li><li>添加到孔中加入250μl更多的PEGDA的，上传L2如上文所述照射L2为60秒。 </li><li>除去未固化的聚合物和洗环异丙醇O 2​​ / N。 </li></ol></li><li>采用静电纺丝可生物降解的PLGA的制备隔膜 注：PEGDA戒指作为模板结束了它的PLGA进行电纺丝与PLGA再现底层的地形，因为它滚动到这些模板。纺丝后，PLGA聚合物的片材从集电极剥离。最终的PLGA膜不含有该被留下附着在金属集电体的PEGDA环。 <ol><li>分布在电镀铝板的PEGDA环（12厘米×20厘米），用于创建一个静态的静电收集器。 </li><li>安装使用导电碳胶带环。请注意：这些一旦形成可以被重新用作用于静电纺丝模板的环。 </li><li>准备纺丝聚合物溶液。溶解的PLGA（50/50 DL-丙交酯（52摩尔％）：乙交酯（48摩尔％），44千克/摩尔）的二氯甲烷（DCM）中的20％w / w的浓度。 </li><li>轰动O 2 / N使用前</li><li>地点4胰岛素注射器（平端，0.8厘米内径针头）在注射泵。注：四个注射器保证比单针筒的工作更快速静电。 </li><li>加载加入2.5ml在每个注射器的PLGA溶液。 </li><li>静电纺丝用30微升/分钟的流速和电压范围从12至15千伏。离开针和15厘米的集电极之间的距离。 </li><li>静电纺丝1小时和30分钟，最后小心剥离从支承PEGDA环集电极的PLGA电纺片材。 </li><li>切静电支架成直径22 mm圆采用圆形打孔和离开位于中心的环结构。 </li></ol></li></ol></li></ol> PLGA微加工膜2。长期储存注：PLGA戒指制作并经认可的外部公司消毒;样品上照射在25-40千戈瑞的外部剂量范围。 <ol><li>装在小容器中的膜（塑料培养皿），并把它放在一个医用级包。 </li><li>使用滤纸来创建小的过滤袋的干燥剂。牛逼o创建袋，折叠滤纸（直径125mm），并削减所产生的半圆一半;然后用胶带封住两端连在一起。 <ol><li>填3滤纸袋用1克二氧化硅橙，钴（II），氯化铜（Ⅱ）硫酸盐，分别。 </li><li>把三袋干燥剂里面的医疗级袋子连同静电膜。 </li></ol></li><li>放入袋子市售6点湿度指示卡检测存储期间的任何水分积聚。 </li><li>使用真空热封机真空并密封袋。 </li><li>发送环膜与外部公司进行γ射线照射。 </li><li>商店γ辐照PLGA膜在很宽的温度范围为-20°C至+37℃，在潮湿的环境中含5％ 二氧化碳培养箱内。 </li><li>交的存储，检查湿度指示器，以确认湿度的水平之下30％。 </li><li> ü本身扫描电子显微镜（SEM）来评估纤维的完整性。 </li></ol> 3，分离角膜缘外植体兔角膜缘外植体兔眼（从农场，兔子繁殖的消费中获得）隔离。 <ol><li>消毒用消毒液（3％）的兔眼。 </li><li>通过消除周围的角膜任何多余的组织清洁眼睛。 </li><li>使用解剖显微镜对角膜的其余的角膜缘区域（标识为角膜（透明）和巩膜（白色）之间的薄的圆形区域）分开。 </li><li>切入解剖显微镜下段（约1.5厘米长）。 </li><li>消毒在1.5％消毒液的角膜缘段1分钟。 </li><li>切缘细分成小块（100-500微米）用手术刀刀片。 </li><li>存储在培养介质中的小片组织（DMEM + Glutamax的：Ham&#39;s F12（1:1），10％胎牛血清，1 U / ml的笔icillin，100毫克/毫升链霉素，2.5微克/毫升的两性霉素，EGF的10纳克/毫升，而胰岛素的5微克/毫升），在37℃和5％CO 2，直到使用（不超过60分钟）。 </li></ol> 4，突增生，从角膜缘外植体细胞兔角膜缘外植体放置在两个新鲜纺丝微制造的膜和薄膜构成，它是真空包装和保存6个月，在-20℃下。 <ol><li>外套环支架用15微升的血纤维蛋白胶：使用细胞刮刀（1 1为18.75毫克/毫升和凝血酶从人血浆中以2.5单位/ ml的浓度的浓度的混合物，从人血浆中的纤维蛋白原），用于分配的纤维蛋白均匀。 </li><li>直接将组织块使用将25g针和解剖显微镜的PLGA micropockets。 </li><li>添加细胞培养基非常轻柔，避免拆卸外植体。 <ol><li>更换介质，每3天（3.7节所描述的媒体配方）和KEEP中培养2周时，在湿润的培养箱中在37℃和5％CO 2。 </li></ol></li><li>固定用3.7％甲醛缓冲液进行10分钟，随后洗涤3次，用PBS中的样品。 </li><li>温育在1微克/毫升的4&#39;染液，6二脒-2 - 苯基吲哚（DAPI）或碘化丙啶（PI）在RT下10分钟。 </li><li>在PBS冲洗3次，存储用铝箔纸覆盖的样本。 </li><li>图片用荧光显微镜在543 nm和800 nm的（双光子）的波长的样品。 </li></ol> 5，设置行动兔角膜受伤的3D模型<ol><li>清洁和上述消毒兔眼。 </li><li>通过浸没于0.14％的氢氧化铵为5分钟，卷绕在兔眼。 </li><li>冲洗眼睛在PBS和刮掉了骨节刀上皮。 </li><li>切开并分离角膜巩膜按钮，清除任何剩余的组织。 </li><li>将角膜上皮面朝下到无菌杯，装满0.5％琼脂的DMEM进行。 </li><li>设置完成后，把角膜上皮面朝上，在小培养皿中，加入培养基（上述配方）到角膜缘区。注意：不要覆盖整个角膜;保持器官培养在气 - 液界面。 </li></ol> 6分离角膜缘外植体和环支架在兔角膜3D模型融入社会<ol><li>涂层具有15微升的血纤维蛋白胶的环膜（1：纤维蛋白原的混合物在1875毫克/毫升和凝血酶以2.5单位/ ml的浓度的浓度）。用细胞刮刀，如上所述。 </li><li>直接将组织块使用将25g针和解剖显微镜的PLGA micropockets。 </li><li>把戒指带在裸露的角膜朝上外植体，在气 - 液界面条件下组织块。 （这些条件先前已有描述22）。 </li><li>维持器官培养模型4周的潮湿培养箱中在37℃和5％CO 2的变化的介质每2天。 </li></ol> 7，评估角膜再生和干细胞维持<ol><li> 4周后，用固定3.7％甲醛的眼角膜。 </li><li>过程中，角膜的组织学传统生产6微米的石蜡切片。 </li><li>染色用苏木精和曙红（H＆E）。 </li><li>对于免疫细胞化学，脱蜡的切片在二甲苯（3分钟）和再水合的100％乙醇（1分钟），70％乙醇（1分钟），和蒸馏水（2分钟）。 </li><li>划定使用Dako公司的笔来界定小区域，避免过度使用抗体的部分。 <ol><li>治疗划定的区域用0.05％胰蛋白酶（100微升）处理20分钟（37℃）。 </li></ol></li><li>用PBS彻底清洗和1小时加入100μl的10％山羊血清（阻塞）。 </li><li>孵育样品与100μl的小鼠单克隆抗体cytokera锡3（CK3）和100微升P63的1％山羊血清在o / n 4℃。 </li><li>用PBS洗涤，并用100微升生物素化的第二抗小鼠抗体（1：1,000在1％山羊血清）中1小时，在室温。 </li><li>添加100μl的FITC-链霉抗（1：100在1％山羊血清），用于在室温30分钟。 </li><li>如上述那样对待该样品用DAPI。 </li><li>图像用荧光显微镜在800纳米（双光子）和488纳米波长下的样品。 </li></ol>

<ol>
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作者什么都没有透露。

这项研究描述了静电膜含在其中微特征的制造及（b）如何准备这类膜用于临床使用的真空包装，γ辐照，然后贮存在使用前（A）的技术。在这个特定的应用程序，我们已经开发出含micropockets的模仿角膜缘干细胞小生境的物理特性PLGA膜。本研究的目的是：（i）描述的方法，为读者提供设计和制作含有微特征的研究棚架成的干细胞小生境组织再生及（ii）提供一个更好地了解读者的贡献所需要的知识如何存储电纺支架的很长一段时间。 在临床应用方面，环膜的存储是极其重要的。在这项工作中，该环的降解进行了研究历时6个月。退化膜是通过水解只须保持无水分的过程中暂停该膜从动左右。黑木等人报告说，通过改变聚乳酸的比例PGA，膜的降解发生变化32。本研究还表明，通过提高PGA的量，电纺膜的降解速率在 ​​体内 22增加。这里已经示出了用真空包装的膜连同一些干燥剂和照射它们，并将它们存储在低温下为6个月，没有在纤维完整性和降解没有发生变化。目前，6个月是因为我们已经研究了用含有micropockets但存储数据这些膜1年已报道在普通静电膜在-20℃下22，现在我们有未公布的数据的储存于-20℃下尽可能2年未降解的迹象。因此，对于长期贮存将被推荐给存储它们干燥，在-206，C，但有可能甚至在印度将它们存储在RT至少6个月（可能更长）。列入湿度指标给出检查包装一直保持膜干在这种情况下，他们将适合为目的的一种简单方法。 从这些环到3D模型角膜细胞的转移micropockets内放置角膜缘外植体时，被证明。该小组最近通过将植在普通PLGA膜（无膜环结构）24日报道转移细胞到体外兔角膜模型。使用本微构支架细胞移植已经采取了一步，因为我们现在特别是微特征中定位组织块。在壁龛内直接将外植体的能力，也可以让医生直接在手术影院避免了洁净室的需要首先扩大角膜缘干细胞使用的膜。虽然这片WOR的k具有集中在用于角膜疾病设备的发展，这种微细加工技术还可以应用于开发了许多其它的应用程序的设备。未来的工作将探索构建体的制造对其它组织如皮肤和骨的再生。 而PEGDA微结构的设计和最初的制造可能是耗时的，一旦制造的结构可以在不降低反复使用多次。因此，PLGA微结构的生物降解膜的通过电纺丝的后续制造可以以相同的速度收集器的装配如下进行，以生产纯（“非结构化”）膜。虽然在本工作中，我们已经使用microstereolit​​hography用于制造模具，其它制造方法，例如三维印刷或注射成型，可以也使用。因此，底层的模具可以由PE制成的出其它的聚合物或金属的，而不是GDA。因此这种技术是非常灵活，研究人员可以很容易地适应方法，以符合自己的需求和设施。 在内部 microstereolithography设置在该研究中使用将不会允许与下30μm的特征的制备构建体;这不是对这里所描述的角膜应用的限制，但它可以是在其它型号的设计是至关重要的。在这种情况下，其他技术如2光子聚合（2PP）可能会感兴趣但是静电纺丝技术可能不允许在亚微米尺度的结构的再现（这是目前正在研究通过我们的组）。 制造过程中的关键步骤是：（i）避免的PEGDA模板，可以通过调整时间和光引发剂的量来控制的overcuring。 （ⅱ）控制电纺丝条件，例如温度和湿度。 （三）妥善保存的electrospu采用真空包装，干燥剂Ñ环膜。 总之，通过在膜的微特征内放置角膜缘组织的外植体，我们已经表明，从上的适当位置区域，小区转移的外植体的细胞生长到兔子受伤角膜和随后的再上皮化的角膜。存储在不同的温度下，膜的降解也进行了研究，并包装协议，它允许长期贮存的膜已被开发出来，后者是在发展中的膜用于临床使用是必不可少的。

角膜，眼睛的缺血性中心外大多数组织，是涉及视力1的最重要的组织之一。有几种类型的细胞维持角膜的​​功能。角膜的顶部最外层包括上皮细胞，可以是在厚度2约5-7层。这层防止细菌侵入到角膜3，并允许氧气4的条目。据报道，该角膜上皮在于龛或隐窝（具有120-150微米大小）在被称为角膜缘5,6角膜的周边区域的干细胞。由于干细胞分裂，子细胞也被称为短暂扩充细胞行驶出龛，并为司继续在细胞移动向心向内和向上导致终末分化细胞在角膜中央区7,8。这些细胞经常擦去眼睛E的眨眼xposing新的细胞下方9。 除了 ​​是上皮干细胞的位置，角膜缘也起着从角膜区域10保持血管的结膜走了作用。损害角膜缘可以通过热/化学烧伤，辐射和也遗传疾病10引起的。当此情况发生时，角膜缘屏障被分解使结膜细胞移动到角膜，血管化的区域，引起疼痛和失明的情况。该情况被称为角膜缘干细胞缺陷（LSCD）10。 不同的天然底物已被报告为可能的干细胞的载体用于辅助在角膜再生。例如，胶原基膜使用Dravida 等 11罗摩和他的同事12日报道在112例患者的研究中使用的纤维蛋白。在治疗的本但是最常用的方法是利用人类羊膜从组织库和文化角膜缘上皮细胞在其表面13,14。一旦单层形成，羊膜粘细胞面朝上到受损的角膜有所有的结膜细胞和疤痕组织从它这个细胞移植14前手术切除。星期内的羊膜降低到几个月留下附着在裸露面积以再生上皮15,16的上皮细胞。该技术已成功地恢复视力但仍然有其临床上限制了它的广泛摄取一些实际问题。由于羊膜是人体组织，它需要进行良好的用组织库的程序被用于对患者的细胞移植前筛查。这种筛选不仅降低了疾病传播的风险，但不能完全消除17。除了这已经有变化的，在p报告羊膜由于跨施主变异18,19和加工方法不同19,20 erformance。除了疾病传播的风险小有用于外科中心能够获得良好的运行组织库，而不是提供给所有的要求。 虽然羊膜是比较成功的，有必要对角膜疾病的治疗的新合成的生物可降解的细胞载体的替代品的开发。综合运营商将克服需要银行程序，以及消除疾病传播和跨捐助变性的风险小。在这个意义上说，材料，如聚乙二醇21,22和PLGA 23,24进行了研究。 在发展中的合成替代羊膜也有设计成其期望的功能，希望帮助培养的细胞的存活的可能性。政府间谈判在生物材料设备微特征为细胞行为的具体控制lusion感兴趣的新兴领域。许多作者报道工作，争取人工干细胞的发展龛25-30。这个小组最近报告创立了微加工PEGDA纤维连接蛋白biofunctionalized人工角膜缘的角膜缘上皮细胞22的交付和含微加工口袋角膜缘上皮细胞31的支持静电的生物降解膜的制作的方法。 这个工作的目的是开发一种新的制造技术用于容纳微特征的模拟的程度，其中干细胞存在于身体的微环境中的生物材料的设备的开发。我们已经开发出结合microstereolit​​hography和静电纺丝的技术，它允许显示格雷亚可生物降解的微结构化的膜的制造中吨潜力的组织再生程序。 注意到，虽然在此工作此技术已经被应用到环角膜再生的制造中，该技术可以应用于器件的制造为各种上皮组织的再生， 例如 ，皮肤，口腔是很重要粘膜，肠，呼吸，和膀胱上皮细胞。具体地说，在该研究中，我们开发了一种合成的生物可降解的膜以相同的方式发挥功能的羊膜来提供细胞对角膜。该膜含有大约300微米micropockets（大于沃格特的Pallisades的角膜缘隐窝（约150微米））。最后，我们建立了封装协议，它允许存储这些膜在-20℃，6个月以上没有显示出崩溃的迹象。

<table border="0" cellpadding="0" cellspacing="0" width="692">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:183px;">Name of&#160; reagents/material/equipment</td>
			<td style="width:173px;">Company</td>
			<td style="width:157px;">Catalogue number</td>
			<td style="width:180px;">Comments (optional)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:183px;">Poly lactic-co-glycolic acid</td>
			<td style="width:173px;">Purac</td>
			<td style="width:157px;">PDLG5004</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:183px;">Dichloromethane</td>
			<td style="width:173px;">Sigma Aldrich Or Fisher</td>
			<td style="width:157px;">270997 Or D/1850/17</td>
			<td style="width:180px;">&#62;99.8% contains 50-150 ppm amylene stabiliser</td>
		</tr>
		<tr height="113">
			<td height="113" style="height:113px;width:183px;">Digital Micromirror Device (DMD) Discovery 1100 Controller Board &#38; Starter Kit</td>
			<td style="width:173px;">Texas Instruments</td>
			<td style="width:157px;">1076N732 (UV)</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">473 nm Laser</td>
			<td style="width:173px;">Laser 2000</td>
			<td style="width:157px;">MBL-III</td>
			<td style="width:180px;">150 mW</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:183px;">Poly (ethylene glycol) diacrylate</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">475629</td>
			<td style="width:180px;">Mn = 250g/mol&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 500 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">DEMEM + Glutamax</td>
			<td style="width:173px;">Fisher</td>
			<td style="width:157px;">12077549</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:183px;">Ham&#8217; s F12</td>
			<td style="width:173px;">Labtech biosera</td>
			<td style="width:157px;">LMH1236/500</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Fetal Bovine Serum</td>
			<td style="width:173px;">Labtech biosera</td>
			<td style="width:157px;">FB-1090/50</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">EGF</td>
			<td style="width:173px;">R&#38;D</td>
			<td style="width:157px;">236-EG-200</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Insulin</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">91077C-1G</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Amphotericin</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">A2942-100ml</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Penicillin/Streptomycin</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">P0781-100ml</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="22">
			<td height="39" rowspan="2" style="height:39px;width:183px;">DAPI</td>
			<td rowspan="2" style="width:173px;">Sigma Aldrich</td>
			<td rowspan="2" style="width:157px;">32670</td>
			<td rowspan="2" style="width:180px;"></td>
		</tr>
		<tr height="17">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Propidium Iodide</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">P4864</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Thrombin</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">T9326</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Fibrinogen</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">F3879</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">p63</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">P3737</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">CK3</td>
			<td style="width:173px;">Merck Millipore</td>
			<td style="width:157px;">CLB218</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Hematoxylin</td>
			<td style="width:173px;">SLS</td>
			<td style="width:157px;">HHS16-500ML</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Eosin</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">HT110232-1L</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:183px;">Medical grade bag (PET/Foil/LDPE) Peelable pouch</td>
			<td style="width:173px;">Riverside Medical Ltd. Derby, UK</td>
			<td style="width:157px;">Foil laminate PET/Foil/LDPE, (12,7,50)</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:183px;">gamma- irradiation (Sterilisation)</td>
			<td style="width:173px;">Applied Sterilisation Technologies (Synergy Health Laboratory Services (SHLS), Abergavenny UK) - external dose range of 25-40KGy</td>
			<td style="width:157px;">N/A</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:183px;">Silica gel orange</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">10087</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:183px;">Cobalt (II) chloride</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">232696</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:183px;">Copper (II) sulphate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:157px;">C-1297</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:183px;">Six spot humidity indicator card</td>
			<td style="width:173px;">SCC, USA</td>
			<td style="width:157px;">6HIC200</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:183px;">Vacuum heat seal machine</td>
			<td style="width:173px;">Andrew James UK Ltd, Bowburn, UK</td>
			<td style="width:157px;">VS518</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:183px;">Andrew James Vacuum Sealer rolls 28cm X 40 metre rolls</td>
			<td style="width:173px;">Andrew James UK Ltd, Bowburn, UK</td>
			<td style="width:157px;">BR2805</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:183px;">Scanning Electron Microscopy (SEM)</td>
			<td style="width:173px;">Philips/FEI XL-20 SEM</td>
			<td style="width:157px;">N/A</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:183px;">Confocal Microscope</td>
			<td style="width:173px;">Zeiss LSM 510 META</td>
			<td style="width:157px;">N/A</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:183px;">Videne Antiseptic Solution</td>
			<td>Ecolab, Swindon, UK</td>
			<td style="width:157px;">N/A</td>
			<td style="width:180px;">3%</td>
		</tr>
	</tbody>
</table>

combination of microstereolithography and electrospinning to produce membranes equipped with niches for corneal regeneration

角膜问题影响全球数百万人减少了他们的生活质量显著。角膜疾病可以由疾病如无虹膜或史蒂芬约翰逊综合症以及由外部因素如化学烧伤或辐射引起的。目前的治疗是（i）使用的角膜移植物以及（ii）利用扩展在实验室和交付的载体（ 例如 ，羊膜）干细胞;这些治疗都是比较成功的，但不幸的是，他们可以经过3-5年的失败。有必要设计和制造能够详细模拟，其中干细胞位于角膜的生理环境新角膜生物材料的设备。角膜缘干细胞存在于被称为沃格特的山花园具体龛（角膜和巩膜之间的圆形区域）的缘。在这项工作中，我们已经开发出一种新的平台技术，该技术结合了两种尖端的制造技术（microstereolit​​hography和electrospinn荷兰国际集团），用于模仿在一定程度上角膜缘角膜膜的制造。我们的膜含有人工micropockets其目的是为细胞提供保护，沃格特的帕利塞德做的眼睛。

静电微细加工环用microstereolithography和静电纺丝的组合（ 图1和图2）制造的。不同尺寸的PEGDA环用microstereolithography（ 图3）制造;此技术允许在制造结构中的厘米的顺序和微特征的同时掺入。在这种情况下，直径为环从1.2-1.6厘米含有350-500微米micropockets制备了（ 图4）。 在制造，杀菌和材料，以便将来临床使用的包装而言，发现在医疗级包装袋的真空包装显著改进以实现长期存储的PLGA膜（ 图5）的能力;使用的医疗级袋（PET /铝箔/聚乙烯），0.12毫米的厚度使我们能够实现较长的保质期。这是通过发送membr调查ANES我们在印度和膜的合作者都在-20℃保存数月内，在室温和37℃，故意潮湿的条件下（潮湿孵化器）， 图5显示了使用存储在无理取闹条件37℃，在潮湿条件下，膜是唯一的稳定约1个月的非真空包装的条件下，但在真空包装条件（ 图5和表1）取得储存3个月。 表1显示了在该下也能选择成有利于水的吸收和纤维溶胀如果注重袋的使用的选择条件来实现存储条件的改善。 环支持细胞生长的不同条件（I）环新鲜制作及（ii）戒指保存6个月（ 图6）角膜缘外植体。小区转移是在4周后达到时复表现很优秀的3D模型受伤的PLGA膜。细胞从组织外植体放置在上创建先前裸露的角膜新的上皮细胞（ 图7），所述膜前身。正（角膜未经任何处理的）和阴性对照（受伤的角膜）也维持培养相同的时间周期。阴性对照证实了在不存在任何添加的细胞的缺乏形成的一种新的上皮细胞。免疫细胞化学证实，细胞从外植体生长出是角膜上皮细胞，因为它们呈阳性反应，角膜分化标记CK3（ 图7E）。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig1highres.jpg" width="500" /> 图1原理图microstereolithography的建立为建立PEGDA环。 her.within页=“总是”&gt; <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig2highres.jpg" width="500" /> 图2原理使用PEGDA微细加工戒指作为模板，静电纺丝的过程。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig3highres.jpg" width="500" /> 图3（A）示出的静态集电极（电镀铝板带PEGDA环），用于微加工的PLGA膜的纺丝的一个例子（B）和（E）示出了不同的静电垫从静态集电极剥离（C）示出了不同尺寸的突出用microstereolithography对于下面的表面的制造的通用性的PEGDA模板（D）示出了PLGA微制造复制品。/files/ftp_upload/51826/51826fig3highres.jpg“目标=”_ blank将“&gt;请点击这里查看该图的放大版本。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig4highres.jpg" width="500" /> àPEGDA环马蹄microfeature（一）图4 SEM图像;精细加工的口袋里（B）的高倍率SEM图像。以下PLGA环马蹄microfeature（C）的相位对比图像;精细加工的口袋（四）高倍率相位对比图像。 <a href="https://www.jove.com/files/ftp_upload/51826/51826fig4highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig5highres.jpg" width="500" /><b温度和时间对存储的真空和非真空的 R /&gt; 图5。影响PLGA封装（各占50％）膜（44千克/摩尔）与微机电环比6个月。膜的完整性被评定为完全不变纤维（+ + +），一些纤维肿胀（+ +），纤维合并（+）或不完整纤维（ - ）。扫描电镜图像和三个干燥剂（硅胶橙色，钴（II），氯化铜（Ⅱ））显示纤维的完整性或湿度无明显变化。 <a href="https://www.jove.com/files/ftp_upload/51826/51826fig5highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig6highres.jpg" width="500" /> 图6荧光图像显示从新鲜出炉的可生物降解的PLGA环角膜缘外植体的LEC的生长（A，B），并于6个月后储存在-20环℃（C，D），图像（A）和（B）对应于细胞用DAPI染色（蓝色）和碘化丙啶（红色）。图像b是共焦Z堆叠的外植体放置于微加工腔的正交视图。图像（C）和（D）表示阳性染色P63（绿色）。 <a href="https://www.jove.com/files/ftp_upload/51826/51826fig6highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig7highres.jpg" width="500" /> 图7（A）中示出了具有位于其上预先涂有纤维蛋白胶支架的环支架和组织外植体兔受伤角膜模型（B）和（C）是阳性和阴性对照。阳性对照是一个新鲜的拉比<html> 。吨角膜和阴性对照角膜其中上皮被故意除去（阴性对照也培养4周）（D）为4周培养后的组织工程角膜的H＆E染色图像;该图中示出了通过将细胞从放置在壁龛外植出来形成的新的多层上皮细胞（E）是免疫细胞图像表示从角膜缘外植体的细胞生长;细胞核用DAPI染色（蓝色）和细胞显示阳性染色细胞角蛋白3，角膜分化标记（绿色）。 <a href="https://www.jove.com/files/ftp_upload/51826/51826fig7highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always" width="611"><tbody><tr height="22"><td height="43" rowspan="2" style="height:43px;width:163px;"> PLGA（50/50） </td><html> X;存储在37℃湿膜“&gt;光纤诚信</td></tr><tr height="21"><td height="21" style="height:21px;width:64px;"> 0天</td><td style="width:64px;"> 1个月</td><td style="width:64px;">第2个月</td><td style="width:64px;"> 3个月</td><td style="width:64px;"> 4月</td><td style="width:64px;"> 5月</td><td style="width:64px;"> 6个月</td></tr><tr height="21"><td height="21" style="height:21px;width:163px;">非真空包装</td><td style="width:64px;"> + + + </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td></tr><tr height="67"><td height="67" style="height:67px;width:163px;">真空（袋）（PE，PA复合材料）厚度：0.14毫米</td><td style="width:64px;"> + + + </td><td style="width:64px;"> + </td><td style="width:64px;"> - </td>64px;“&gt; - </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td></tr><tr height="61"><td height="61" style="height:61px;width:163px;">真空（袋乙）（PET /铝箔/聚乙烯）厚度：0.75毫米</td><td style="width:64px;"> + + + </td><td style="width:64px;"> + + + </td><td style="width:64px;"> + + + </td><td style="width:64px;"> + </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td></tr></tbody></table> 真空和不同的储物袋上的PLGA诚信表1的影响（各占50％）膜（44千克/摩尔）检查6个月以上的存储空间。膜的完整性被评定为完全完整纤维（+ + +），一些纤维肿胀（+ +），纤维合并（+）或不完整纤维（ - ）。

Watch this Scientific Journal Video about Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration at JoVE.com

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

我们报告的静电膜，用来研究细胞行为中micropockets的制造技术。具体地，我们描述了用于生产的PLGA（聚（丙交酯 - 共 - 乙））配备有微特征角膜生物材料的设备microstereolit​​hography和静电纺丝的组合​​。

Microstereolit​​hography和静电结合，以产生膜配备有利基的角膜再生

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

combination-microstereolithography-electrospinning-to-produce

Research

JoVE Journal

Bioengineering

12.4K 次观看.  University of Sheffield. 我们报告的静电膜，用来研究细胞行为中micropockets的制造技术。具体地，我们描述了用于生产的PLGA（聚（丙交酯 - 共 - 乙））配备有微特征角膜生物材料的设备microstereolit​​hography和静电纺丝的组合​​。 

视频: Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

静电基础：优化解决方案和设备参数

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

科研

生物工程

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

人类血管周围干细胞用于骨再生

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

通过结合表面接枝和修改后的过程光敏感膜的制备

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

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. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

适应静电过程中提供三个独特的环境为一个三层次<em&gt;在体外</em&gt;的气道壁模型

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Merging Ion Concentration Polarization between Juxtaposed Ion Exchange Membranes to Block the Propagation of the Polarization Zone

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP induces a noninvasive region for charged biomolecules (i.e., the ion depletion zone), and targets can be preconcentrated on this region boundary. Despite the high preconcentration performances with ICP, it is difficult to find the operating conditions of non-propagating ion depletion zones. To overcome this narrow operating window, we recently developed a new platform for spatiotemporally fixed preconcentration. Unlike preceding methods that only use ion depletion, this platform also uses the opposite polarity of the ICP (i.e., ion enrichment) to stop the propagation of the ion depletion zone. By confronting the enrichment zone with the depletion zone, the two zones merge together and stop. In this paper, we describe a detailed experimental protocol to build this spatiotemporally defined ICP platform and characterize the preconcentration dynamics of the new platform by comparing them with those of the conventional device. Qualitative ion concentration profiles and current-time responses successfully capture the different dynamics between the merged ICP and the stand-alone ICP. In contrast to the conventional one that can fix the preconcentration location at only ~5 V, the new platform can produce a target-condensed plug at a specific location in the broad ranges of operating conditions: voltage (0.5-100 V), ionic strength (1-100 mM), and pH (3.7-10.3).

The protocol for a novel ion concentration polarization (ICP) platform that can stop the propagation of the ICP zone, regardless of the operating conditions is described. This unique ability of the platform lies in the use of merging ion depletion and enrichment, which are two polarities of the ICP phenomenon.

并列的合并离子交换膜之间的离子浓差极化，以块极化区的传播

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水凝胶的合成与防污性能作为膜的净水

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

用于组织工程应用的三维聚 (ε-内) 支架的熔体静电纺丝

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Microstereolit​​hography和静电结合，以产生膜配备有利基的角膜再生

Microstereolithography和静电结合，以产生膜配备有利基的角膜再生