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. 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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</su...

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）。...

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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 ...

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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。 