大昌行，MB和FK是由EPSRC按照有关基金的支持。 BL-V和DS分别支持MRC博士助学金。 KC是由来自英国的再生医学平台提供资金支持。

1，合成PHNGAD（聚[1,6-hexanodiol /新戊二醇/二（乙二醇）-alt - 己二酸]二醇）的<img alt="方案1" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme1.jpg" width="500" /> 方案1:PHNAGD合成 PHNAGD的合成的示意图。 PHNAGD制备的1,6-Hexanodiol的反应中，二甘醇，neoppentyl乙二醇和己二酸。 PHNAGD，聚[1,6-hexanodiol /新戊二醇/二（乙二醇）-alt - 己二酸]二醇。 <ol><li>适用于热处理的单体1,6 - 己二醇，二（乙二醇）和新戊二醇，在40℃下进行48小时的真空炉中，以除去任何残余的水。允许冷至室温下真空。 </li><li>加入22毫摩尔的各单体和己二酸（55毫摩尔）到两颈圆底烧瓶配有搅拌棒并连接到一个Dean-Stark装置。 </li><li>放置在真空下整机组装，轻轻加热GLAssware在40℃下6小时，以避免在加入化学品向烧瓶中的任何湿气的吸收。 </li><li>添加经由注射器，一滴一滴，0.0055摩尔的催化剂的钛（Ⅳ）丁醇。 </li><li>搅拌该反应混合物在180℃下，N 2气氛下进行24小时，并在Dean-Stark分水器收集残留的水。使产物冷却至室温。 </li></ol> 2，合成PU134的<img alt="方案2" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme2.jpg" width="500" /> 方案2:PU134合成聚氨酯134的PU134的制备是在1.0当量ÂPHNGAD与2.0当量4,4&#39;--亚甲基双（异氰酸苯酯）的反应合成的示意图，接着加入1.0。当量的1,4 - 丁二醇扩链剂。 <ol><li>混一当量的多元醇PHNGAD的（锰〜1800道尔顿，3.2毫摩尔）与两个当量的4&#39;4-亚甲基双（苯基异氰酸酯）（6.4毫摩尔）在无水N，N二甲基甲酰胺（12毫升）中。 </li><li>搅拌反应混合物，在70℃，N 2气氛下。 </li><li>添加经由注射器逐滴催化剂的钛（Ⅳ）叔丁醇钾（0.8％重量）。 </li><li> 1小时后，加入一当量的增链剂1,4丁二醇（3.2毫摩尔）。增加，在90℃的温度和N 2气氛下搅拌24小时。 </li><li>以下的反应中，（也可以用己烷或水），加入乙醚收集的聚氨酯通过沉淀，直至出现沉淀逐滴加入到反应溶液中。 </li><li>离心溶液以5300×g离心5分钟。 </li><li>倒出上清液并擦干，在40℃的真空烘箱中，直到溶剂蒸发。 注意:为了确保反应的最终产物具有对于对分子量分布的正确参数，功能GROU的聚合物，熔点和玻璃化转变温度的PS，各种分析技术和方法都可以使用，如凝胶渗透色谱法或FITR光谱。 </li></ol> 3，准备PU134解决方案<ol><li>称取200毫克PU134到一个玻璃瓶。 </li><li>比为1:四氢呋喃，及组合四氢呋喃和dicloromethane在1比1，稀释:PU134到2％的终浓度在许多溶剂:氯仿，氯仿和甲苯中的1的组合。 注意:根据要使用的聚合物上的合适的溶剂的选举而变化。不同的溶剂具有不同的沸点，可以影响聚合物的溶解。 </li><li>摇使用振动器的溶液剧烈20分钟，在室温以200 MOT /分钟的速度，直到溶液变得均匀，没有沉淀物观察到的。 </li></ol> 4，镀膜玻璃幻灯片与PU134的<ol><li>将一个柔第二15毫米2盖玻片上的旋涂机。 </li><li>适用于50微升的PU134的解决方案给每个使用吸液管。调整PU134溶液的体积相应于所要求的盖玻片大小保持表面比成比例的量。 </li><li>旋转每个盖玻片7秒，23×g的。 </li><li>空气干燥的盖玻片，在室温下灭菌前至少24小时。 </li></ol> 5，辐射盖玻片<ol><li>通过将10灰色的剂量使用实验室照射12分钟伽玛辐照聚合物涂层的盖玻片。 </li><li>紫外用30瓦紫外线灯16分钟每侧照射聚合物涂层的盖玻片。 </li><li>根据滑动大小将聚合物涂覆的盖玻片在合适的组织培养板中。 </li></ol> 6，扫描电子显微镜<ol><li>通过在5×10 -1毫巴的压力的气氛中溅射为200秒黄金涂层聚合物的盖玻片。 </li><li>捕捉微克使用扫描电子显微镜以20千伏在二次电子成像模式的加速电压，聚合物涂覆的盖玻片的raphs。 </li></ol> 7，原子力显微镜观察<ol><li>扫描20的聚合物表面的×20微米的区域。 </li><li>设置的扫描率从1.32 Hz至1.60赫兹。 </li><li>设置在扫描区域为512×512像素的分辨率。 </li><li>计算均方根涂层的方（RMS或RQ）通过利用从平均影像数据平面截取的高度偏差的平均值，表示为: <img fo:content-width="1in" src="/files/ftp_upload/51723/51723eq1.jpg" width="123" /> 其中Z是当前Z值，而N是在给定区域内的点的数量。 </li><li> 7.5计算的偏差或平均表面粗糙度（Ra）使用图像的 <img fo:content-width="1.5in" src="/files/ftp_upload/51723/51723eq2.jpg" width="154" /> 其中Z（x）是一个描述吨的功能他表面轮廓的高度（Z）和该样品在评价长度"L"的位置（x）来分析。 Ra表示的表面相对于中心面的平均值。 </li></ol> 8，细胞培养和分化<ol><li>培养并如干草17所述的分化的人类胚胎干细胞系（人类胚胎干细胞）H9。 </li><li>分离用的解离试剂的细胞和replate他们到PU134包被的载玻片，在分化过程的第9天，在无血清培养基中的存在，作为描述在Szkolnicka 18,19。 注意:使用的酶的细胞解离的优选物理小区脱离。 </li></ol> 9，细胞色素P450功能分析<ol><li>测量CYP3A活性按照制造商的说明进行操作。 </li><li>培育人类胚胎干细胞衍生的肝细胞在13日或19日，和媒体没有细胞 - 作为阴性对照，用适当的SUBSTR连吃5小时，在37℃。 </li><li>收集细胞和培养基的上清液，进行该测定按照生产商的说明进行操作。 </li><li>测量CYP活性和归一化的相对的表面面积（cm 2）的水平。 </li></ol> 10，免疫染色<ol><li>洗涤胚胎干细胞衍生的肝细胞用PBS，1分钟，重复2次。 </li><li>添加冰冷的100％甲醇中固定细胞，置于-20℃下进行10分钟。 </li><li>用PBS进行5分钟清洗细胞，并重复两次。 </li><li>孵育细胞用PBS / T（0.1％吐温）/ 10％BSA进行1小时，在室温。 </li><li>吸出的PBST溶液并加入稀释在PBS / T（0.1％吐温）/ 1％BSA中的相应的第一抗体，孵育在4℃下温和搅拌O 2 / N。 </li><li>洗涤细胞，用PBS / T（0.1％吐温）/ 1％BSA中进行5分钟，重复三次。 </li><li>稀释在PBS / T（0.1％吐温）/ 1％BSA中的相应抗体，加入到细胞中，并孵育在黑暗中在RT下1小时，轻轻摇动。 </li><li>用PBS进行5分钟清洗细胞，并重复三次。 </li><li>添加MOWIOL 488（含DAPI 1:1,000）到每个孔中，加盖玻片轻轻以减少气泡的数目。储存固定的细胞在4°C黑暗。观察用显微镜适当的过滤器和荧光灯染色。优化的第一和第二抗体都列在表1中 。 </li></ol>

<ol>
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J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126 (4), 677-689 (2006).</li><li>Dang, J. M., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+Induction+of+Aligned+Mesenchymal+Stem+Cell+Sheets+by+Culture+on+Thermally+Responsive+Electrospun+Nanofibers.">Myogenic Induction of Aligned Mesenchymal Stem Cell Sheets by Culture on Thermally Responsive Electrospun Nanofibers.</a> Advanced Materials. 19 (19), 2775-2779 (2007).</li><li>Azevedo, E. C., Nascimento, E. M., Chierice, G. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV+and+gamma+irradiation+effects+on+surface+properties+of+polyurethane+derivate+from+castor+oil.">UV and gamma irradiation effects on surface properties of polyurethane derivate from castor oil.</a> Pol&#237;meros. 23 (3), 305-311 (2013).</li><li>Rosu, L., Cascaval, C. N., Ciobanu, C., Rosu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+UV+radiation+on+the+semi-interpenetrating+polymer+networks+based+on+polyurethane+and+epoxy+maleate+of+bisphenol+A.">Effect of UV radiation on the semi-interpenetrating polymer networks based on polyurethane and epoxy maleate of bisphenol A.</a> Journal of Photochemistry and Photobilogy A: Chemistry. 169 (2), 177-185 (2005).</li><li>Yang, X. F., Tallman, D. E., Bierwagen, G. P., Croll, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blistering+and+degradation+of+polyurethane+coatings+under+different+accelerated+weathering+tests.">Blistering and degradation of polyurethane coatings under different accelerated weathering tests.</a> Polymer Degradation and Stability. 77 (1), 103-109 (2002).</li></ol>

大昌行是民间组织，董事，创始人和FibromEd制品有限公司MB和JPI股东是公司创始人股东FibromEd制品有限公司

许多用于产生从干细胞的肝细胞的现有方法依赖于动物来源的未定义的矩阵。这些基材可以是昂贵的，充满变数，影响细胞的功能和稳定性，较显著障碍中的应用。因此，我们进行了一个屏幕的量支持的干细胞衍生的肝细胞培养的合成材料。我们已经确定了，一个简单的聚氨酯（PU134），通过聚合PHNGAD，MDI和一个扩展，在结合强大的肝细胞分化的方式形成，稳定肝细胞的表型，当与基质胶15相比提高了细胞的功能。 处理现有的生物基质，培养中常用的分化干细胞，如玻连蛋白22，层粘连蛋白521 23或基质胶的，是有效的短期内维修。相比之下，PU134涂层表面提供了优越的底hepatocy德文化。此外，表面形貌与聚合物灭菌的优化导致了电池的性能，在提供人类肝模型在规模和可靠的性能的关键因素进一步改进。 扩链剂与异氰酸苯酯的性质表示该过程的关键步骤之一;为消除或更换任何部件会显著损害聚合物表面的，因此，细胞附着和干细胞衍生的肝细胞功能活性。该聚合物的组合物将影响物理参数，如弹性和湿润性24，其具有影响聚合物的吸收涉及肝功能键外基质蛋白的能力。此外，不同的催化剂，反应时间和温度会影响聚合物的分子量分布。 所用溶剂的选择，以溶解该聚合物重呈现在一个优化的聚合物涂覆的表面的取得另一个临界点。我们已经观察到所用溶剂的性质影响涂布的表面，其具有在细胞25-31的功能直接影响的地形。另外，纺丝时和速度，可以在取得此均匀和均一的涂层是至关重要的。另一个步骤采取在考虑是适用于聚合物涂覆表面的灭菌过程，因为细胞的活性可以通过使用该杀菌处理的影响。这可能是因为敏感区域（S）的聚合物，以不同的灭菌程序32-34的结构内的存在。 目前的多能性干细胞的基础细胞模型具有表型和功能上的限制。我们相信，这个优化的聚合物涂覆的表面代表在本领域的进步，绕过一些与使用生物SUBST相关联的局限性率。此外，使用内三维聚合物的可能性矩阵，以支持在感兴趣的组织中发现的不同的细胞类型的附接，可以进一步提高电池的保真度。 最后，该合成材料的使用正变得在从多能干细胞的递送人的体细胞中越来越重要。我们已经开发了一种优化的表面和涂层的过程，是稳健和可靠的传送功能的人类肝细胞为基础的细胞模型和再生医学显著影响。

生物材料已经被广泛用于多能干细胞1的维持和分化。同时使这些生物基质通常包含未定义了大量元器件。基质胶是一种常用的基质干细胞的培养和分化。不幸的是，它的可变成分影响细胞功能和表型。虽然各种各样的替代方案中，更明确的生物基质已用于2-7，其动物来源或可扩展性差，使它们不适合的候选用于工业制造。因此合成的替代品，具有固定的成分，性能可靠的鉴定，是干细胞研究的主要目标。 在克服不确定的细胞培养基质的限制的尝试，化学和生物学的交叉学科的合作已经确定了合成材料，以支持细胞表型的能力。合成器客位基板具有可扩展性，成本效益和可制造成复杂的三维结构，模仿体内环境。由于这些特性的合成底物已被广泛使用，以支持和驱动许多细胞类型8-10的分化。 先进的高通量检测提供了便利合成材料的快速筛选，从大型图书馆，并提供新的材料，具有灵活的特性，在生物医药研究和开发11-13广泛的应用。利用高通量，聚合物微阵列筛选技术，我们迅速确定了一个简单的聚氨酯（PU134），适用于维护人类干细胞衍生的肝细胞。该聚合物被认为是优于动物源性底物对于肝细胞的分化和功能14-16。我们随后优化的涂覆条件，地形和灭菌过程来访问效果在稳定肝细胞的功能和寿命聚合物的性能。这有针对了解肝细胞生物学细胞建模和再生医学应用基础显著影响。 此处所描述的技术代表了如何由合成聚合物的表面可以进行优化，以保持细胞的表型的实例。我们认为，这种技术提供了高效的无血清肝细胞分化方案的组合有可能提供一种可伸缩的生产肝细胞在体外模型和再生医学中使用。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Synthesis, preparation, coating and characterization of polymer PU134 coated coverslips</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Edmun B&#252;hler</td>
			<td>KS-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Irradiator</td>
			<td>CIS Biointernational</td>
			<td>IBL 637&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Specialty Coating System</td>
			<td>&#160;P-6708</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope&#160;</td>
			<td>Philips</td>
			<td>XL30CPSEM</td>
			<td></td>
		</tr>
		<tr>
			<td>Atomic Force Microscope</td>
			<td>DimensionV Nanoscope, VEECO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p4-GLO CYP3A4</td>
			<td>Promega</td>
			<td>V8902</td>
			<td></td>
		</tr>
		<tr>
			<td>UV bulb</td>
			<td>ESCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoScope analysis software</td>
			<td>VEECO</td>
			<td></td>
			<td>version 1.20</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Olympus</td>
			<td>TH45200</td>
			<td>Use Volocity 4 Software</td>
		</tr>
		<tr>
			<td>Tissue culture plates</td>
			<td>Corning, UK&#160;</td>
			<td>3527</td>
			<td></td>
		</tr>
		<tr>
			<td>glass slides</td>
			<td>Scientific Laboratory Supplies</td>
			<td>MIC3308</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethylene glycol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>93171</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;1,6-hexanediol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>240117</td>
			<td></td>
		</tr>
		<tr>
			<td>Neopentyl glycol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>408255</td>
			<td></td>
		</tr>
		<tr>
			<td>Adipic acid</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>9582</td>
			<td></td>
		</tr>
		<tr>
			<td>anhydrous N,N-Dimethylformamide</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>676845</td>
			<td></td>
		</tr>
		<tr>
			<td>titanium (IV) butoxide&#160;</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>244112</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-butanediol&#160;</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>493732</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum oven</td>
			<td>Thermoscientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4,4&#8217;-Methylenebis(phenyl isocyanate)</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>101688</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrahydrofurane</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>401757</td>
			<td></td>
		</tr>
		<tr>
			<td>Sputter coater</td>
			<td>Bal-Tec SCD 050</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Inmunostaining</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline (-MgCl2, -CaCl2)</td>
			<td>Gibco</td>
			<td>10010031</td>
			<td>&#160;Store at room temperature</td>
		</tr>
		<tr>
			<td>PBST, PBS made up with 0.1% TWEEN 20&#160;</td>
			<td>&#160; Scientific Laboratory Supplies Ltd</td>
			<td>EC607&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>&#160; Scientific Laboratory Supplies Ltd</td>
			<td>CHE5010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich, UK</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>MOWIOL 488 DAPI</td>
			<td>Calbiochem</td>
			<td>475904</td>
			<td>Made up in Tris HCL and glycerol as per manufacturers instructions</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cell culture and Functional assay</td>
		</tr>
		<tr>
			<td>CYP3A activity pGLO kit</td>
			<td>Promega</td>
			<td>V8902</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepatozyme</td>
			<td>Gibco</td>
			<td>17705021</td>
			<td></td>
		</tr>
		<tr>
			<td>TryLE express</td>
			<td>Life Technologies</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/12604013">12604013</a></td>
			<td></td>
		</tr>
	</tbody>
</table>

stabilizing hepatocellular phenotype using optimized synthetic surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

聚合物溶剂影响聚合物涂覆的表面的形貌 聚氨酯134溶解在氯仿中，无论是单独使用或与甲苯或四氢呋喃或二氯甲烷中，并在载玻片上旋涂一层的不同制剂的组合。扫描电子显微镜（SEM）和原子力显微镜（AFM）进行了表征的聚合物涂层的物理性能（ 图1）。使用甲苯或氯仿中得到的涂层不是均匀的，这表明一个不均匀的涂层与PU134沉淀（ 图1A）。与此相反，四氢呋喃是一种合适的溶剂，允许一个更均匀的表面（ 图1A）的旋涂。表面形貌是使用均方根（RMS），平均粗糙度（Ra）用AFM测得的。 PU134溶于四氢呋喃展出粗糙度的降低相比40％为the其他溶剂（ 图1B和1C）。这些数据表明，使用四氢呋喃作为溶剂，提供PU134的生物应用的更均匀的涂层。 聚合物形貌对肝功能的影响显著 人类胚胎干细胞，可以高效率地分化为肝脏内胚层在体外表现出许多在成年肝17-20发现的功能。肝脏内胚层的分化诱导，并在第9天成肝细胞样细胞是明显的，其中大部分细胞表达细胞角蛋白19（99％±1.2），α-胎蛋白（99％±0.6），和肝细胞核因子4α（97％±的2.6）;和白蛋白的水平低（20％±1.7）（ 图2）。在这一点上，将细胞用的TrypLE表达和再接种到不同聚合物涂覆的表面脱离。作为代谢功能的量度，细胞色素P450˚F恩膏评估后4天replating。我们观察到在细胞再接种于四氢呋喃/ PU134涂层的表面（ 图3）的2倍，增加的代谢活性。这些结果表明，人类胚胎干细胞衍生的肝细胞代谢活性与PU134的更均匀的涂层的提高，这是与以前的方法21。 关于该聚合物的生物活性性质的表面灭菌的影响 还研究了灭菌的上PU134性能的影响。聚合物涂覆的玻璃载片暴露于UV或γ射线照射。中PU134涂层玻片相衬成像均无差异总额张贴紫外线或γ射线（ 图4A）。这些观察通过扫描电镜分析（ 图4B）被进一步证实。使用人类胚胎干细胞衍生的肝细胞在后10天replating PU134的生物学性能进行了检查。人类胚胎干细胞衍生的肝细胞铺板在γ-辐射PU134涂层的玻璃载片显示了细胞再接种于紫外线辐射PU134涂覆的玻璃载片（ 图4C）的3倍，增加CYP3A活性。这些观察结果表明，γ射线照射，对于我们而言最佳的灭菌技术。 <img alt="图1" src="/files/ftp_upload/51723/51723fig1highres.jpg" /> 图1中优化的聚氨酯134涂覆的表面的（A）玻璃载玻片上涂布PU134的2％的溶液溶解在不同的溶剂中。不同的涂覆的玻璃载片的扫描电子显微（SEM）图像显示，当使用四氢呋喃，用溶剂（黑色和白色箭头）的甲苯，氯仿或它们的混合物比较均匀的涂布面的存在与不存在聚合物的沉淀。这些照片被采取x1664放大和规模的酒吧AR<html> Ë显示的图像下方。 （BC）的原子力显微镜（AFM）是用来分析在选定溶剂中的PU134涂层表面的粗糙度。的粗糙度参数的RMS（均方根）（B）和不同PU134涂层的表面Ra为（平均粗糙度），（C）的分析表明，通过使用四氢呋喃作为溶剂，所得到的涂层具有光滑的表面，当与比较的条件其余组（n = 7）。数据表示为平均值±标准差，P &lt;0.05表示为*，P &lt;0.01表示**， 和 p &lt;0.001表示***，通过学生t检验，相比于载玻片涂有PU134溶解在四氢呋喃测定。误差条代表1个标准差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51723/51723fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> AYS"&gt; <img alt="图2" src="/files/ftp_upload/51723/51723fig2highres.jpg" /> 图2表征的人类胚胎干细胞源性肝细胞中，肝标志物AFP，CK19，HNF4α和白蛋白的人类胚胎干细胞（H9亚型）免疫组化显示表达式得出肝胚层。阴性对照进行与相应的免疫球蛋白G（IgG）的和有代表性的图像被显示。阳性细胞和标准偏差的百分比从视图包含每个视图的至少300个细胞至少五个随机场进行估计。这些照片被拍摄时放大20倍。数据表示为平均值±标准差误差条代表一个标准差。 HNF4α，肝细胞核因子4α;法新社报道，α-胎儿蛋白;白蛋白，白蛋白;细胞角蛋白19，细胞角蛋白19，IgG羊，免疫球蛋白G抗羊，鼠IgG抗体，免疫球蛋白G抗鼠。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51723/51723fig2highres.jpg" target="_blank">请点击这里查看大图versi</a>在这个数字的。 <img alt="图3" src="/files/ftp_upload/51723/51723fig3highres.jpg" /> 图中PU134涂层表面的形貌上的人类胚胎干细胞衍生的肝细胞的功能3功能的影响。保持96小时在载玻片上涂布有PU134溶解在不同溶剂中的hESC衍生的肝细胞中的细胞色素P450 3A活性的测定。上保持在PU134涂层玻璃载玻片细胞中的改进，在细胞色素的活性溶解，用四氢呋喃（N = 6）。数据表示为平均值±标准差，P &lt;0.05表示为*，P &lt;0.01表示**， 和 p &lt;0.001表示***，由学生t检验比较测量，以保持在载玻片上涂布有人类胚胎干细胞衍生的肝细胞PU134溶解在四氢呋喃。误差棒表示1个标准差。 图4优化涂层表面的灭菌。相衬图像（A）或SEM图像（B）表示无毛的差异在γ-辐射或紫外线辐射（UV）处理之间的PU134涂层表面。相位对比图像拍摄于40倍的放大倍率和扫描电镜图像在1,664X放大。 （ 三）分析保持在PU134涂层载玻片10天对人类胚胎干细胞源性肝细胞色素P450 3A活性。在细胞铺板在γ-辐射PU134涂层的玻璃载片，与细胞再接种于紫外线相比，在细胞色素P450 3A活性的增加辐射PU134涂层的玻璃载片。活性单位表示为相对光单位毫升-1 cm -2的培养表面（RLU）组（n = 3）。数据表示为平均值±标准差，P &lt;0.001ìš记***通过学生t检验，相比于保持在由γ-射线消毒PU134涂覆的玻璃盖玻片上的hESC衍生的肝细胞中测量的。误差条代表1个标准差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51723/51723fig4highres.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"><tbody><tr><td>一抗</td><td></td><td></td><td></td></tr><tr><td>抗原</td><td>类型</td><td>公司</td><td>稀释</td></tr><tr><td> HNF4α </td><td>兔多克隆</td><td>圣克鲁斯</td><td> 1/100 </td></tr><tr><td>白蛋白</td><td>鼠单克隆</td><td>西格玛</td><td> 1/500 </td></tr><tr><td>法新社</td><td>鼠单克隆</td><td> Abcam公司</td><td> 1/500 </td></tr><tr><td>细胞角蛋白19 </td><td>鼠单克隆</td><td> Dako公司</td><td> 1/50 </td></tr><tr><td>抗体</td><td>鼠单克隆</td><td> Dako公司</td><td> 1/500 </td></tr><tr><td>二抗</td><td></td><td></td><td></td></tr><tr><td>抗兔568 Alexa的面粉</td><td>山羊</td><td> Invitrogen公司</td><td> 1/400 </td></tr><tr><td>抗鼠488 Alexa的面粉</td><td>兔子</td><td> Invitrogen公司</td><td> 1/500 </td></tr></tbody></table> 表1列出本研究中使用优化的初级和二级抗体和浓度 。 HNF4α，肝细胞核因子4α;法新社报道，α-胎儿蛋白; IgG抗体，免疫球蛋白G。

Watch this Scientific Journal Video about Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces at JoVE.com

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

本文将重点发展聚合物涂层表面进行长期，干细胞的培养稳定来源的人肝细胞。

稳定肝细胞表型采用优化合成表面

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing ...

stabilizing-hepatocellular-phenotype-using-optimized-synthetic

Nanyang Technological University

Research

JoVE Journal

Chemistry

10.2K 次观看.  University of Edinburgh. 本文将重点发展聚合物涂层表面进行长期，干细胞的培养稳定来源的人肝细胞。 

视频: Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

BMP-2的共价结合利用表面上的自组装单层膜法

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

科研

化学

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.

The objective of this research was to form synthetic plant cell wall tissue using layer-by-layer assembly of nanocellulose fibrils and isolated lignin assembled from dilute aqueous suspensions.&#160; Surface measurement techniques of quartz crystal microbalance and atomic force microscopy were used to monitor the formation of the polymer-polymer nanocomposite material.

朝仿生学木:纳米纤维素，木质素的装配式自由站立电影和合成聚阳离子

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and ...

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3]2+ (bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of novel materials. Coupled with analysis by in situ secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS), soft landing provides unprecedented insights into the interactions of well-defined species with surfaces.

在质谱选择离子软着陆准备好定义的表面原位二次离子质谱和红外光谱

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

合成方法通过固相树脂为基础的方法不对称二茂铁产生的生物共轭系统

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional units (e.g., domains). Traditional consensus-building models fail to account for interpositional dependencies &#8211; functionally required covariation of residues that tend to appear simultaneously throughout evolution and across the phylogentic tree. These relationships can reveal important clues about the processes of protein folding, thermostability, and the formation of functional sites, which in turn can be used to inform the engineering of synthetic proteins. Unfortunately, these relationships essentially form sub-motifs which cannot be predicted by simple &#8220;majority rule&#8221; or even HMM-based consensus models, and the result can be a biologically invalid &#8220;consensus&#8221; which is not only never seen in nature but is less viable than any extant protein. We have developed a visual analytics tool, StickWRLD, which creates an interactive 3D representation of a protein alignment and clearly displays covarying residues. The user has the ability to pan and zoom, as well as dynamically change the statistical threshold underlying the identification of covariants. StickWRLD has previously been successfully used to identify functionally-required covarying residues in proteins such as Adenylate Kinase and in DNA sequences such as endonuclease target sites.

Synthetic protein sequences based on consensus motifs typically ignore co-evolving residues, that imply interpositional dependencies (IPDs). IPDs can be essential to activity, and designs that disregard them may result in suboptimal results. This protocol uses StickWRLD to identify IPDs and help inform rational protein design, resulting in more efficient results.

优化合成蛋白质:Interpositional依赖性的鉴定指示在结构和/或功能性连接的残留

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

一个优化的协议通过使用金纳米粒子的放射性标记效率<sup&gt; 125</sup&gt;我标记的叠氮辅基

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Color Spot Test As a Presumptive Tool for the Rapid Detection of Synthetic Cathinones

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and other border protection agencies globally. Color testing is a presumptive identification technique indicating the presence or absence of a particular drug class using rapid and uncomplicated chemical methods. Owing to their relatively recent emergence, a color test for the specific identification of synthetic cathinones is not currently available. In this study, we introduce a protocol for the presumptive identification of synthetic cathinones, employing three aqueous reagent solutions: copper(II) nitrate, 2,9-dimethyl-1,10-phenanthroline (neocuproine) and sodium acetate. Small pin-head sized amounts (approximately 0.1-0.2 mg) of the suspected drugs are added to the wells of a porcelain spot plate, and each reagent is then added dropwise sequentially before heating on a hotplate. A color change from very light blue to yellow-orange after 10 min indicates the likely presence of synthetic cathinones. The highly stable and specific test reagent has the potential for use in the presumptive screening of unknown samples for synthetic cathinones in a forensic laboratory. However, the nuisance of an added heating step for the color change result limits the test to laboratory application and decreases the likelihood of an easy translation to field testing.

Here we present a simple, inexpensive, and selective chemical spot test protocol for the detection of synthetic cathinones, a class of new psychoactive substances. The protocol is suitable for use in various areas of law enforcement that encounter illicit material.

色斑试验作为合成 Cathinones 快速检测的推定工具

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

基质辅助激光解吸电离飞行时间 (MALDI) 质谱法表征合成聚合物

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

Light-driven Molecular Motors on Surfaces for Single Molecular Imaging

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces are demonstrated. This work requires careful design, considerable synthetic effort, and proper analysis. The rotary motion of the molecular motor in solution is shown by 1H NMR and UV-vis absorption spectroscopy techniques. In addition, the method to graft the motor onto an amine-coated quartz is described. This method helps to gain more insight into molecular machines.

The manuscript describes how to synthesize and graft a molecular motor on surfaces for single molecular imaging.

表面光驱动分子电机的单分子成像

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces ...

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

大肠杆菌无细胞蛋白合成: 一种强大、灵活和可访问的平台技术的协议

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...