Name: stabilizing hepatocellular phenotype using optimized synthetic surfaces
Uploaded: 2014-09-26T14:00:00.000+00:00
Duration: 8 min 50 s
Description: Watch this Scientific Journal Video about Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces at JoVE.com

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>

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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</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><tbody><tr><td colspan="4">&lt;strong&gt;Synthesis, preparation, coating and characterization of polymer PU134 coated coverslips&lt;/strong&gt;</td></tr><tr><td>Shaker</td><td>Edmun B&amp;uuml;hler</td><td>KS-15</td><td></td></tr><tr><td>Irradiator</td><td>CIS Biointernational</td><td>IBL 637&amp;nbsp;</td><td></td></tr><tr><td>Spin coater</td><td>Specialty Coating System</td><td>P-6708</td><td></td></tr><tr><td>Scanning Electron Microscope&amp;nbsp;</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&amp;nbsp;</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&amp;ndash;Aldrich</td><td>93171</td><td></td></tr><tr><td>1,6-hexanediol</td><td>Sigma&amp;ndash;Aldrich</td><td>240117</td><td></td></tr><tr><td>Neopentyl glycol</td><td>Sigma&amp;ndash;Aldrich</td><td>408255</td><td></td></tr><tr><td>Adipic acid</td><td>Sigma&amp;ndash;Aldrich</td><td>9582</td><td></td></tr><tr><td>anhydrous &lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide</td><td>Sigma&amp;ndash;Aldrich</td><td>227056</td><td></td></tr><tr><td>Diethyl ether</td><td>Sigma&amp;ndash;Aldrich</td><td>676845</td><td></td></tr><tr><td>titanium (IV) butoxide&amp;nbsp;</td><td>Sigma&amp;ndash;Aldrich</td><td>244112</td><td></td></tr><tr><td>1,4-butanediol&amp;nbsp;</td><td>Sigma&amp;ndash;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&amp;rsquo;-Methylenebis(phenyl isocyanate)</td><td>Sigma&amp;ndash;Aldrich</td><td>101688</td><td></td></tr><tr><td>Tetrahydrofurane</td><td>Sigma&amp;ndash;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 colspan="4">&lt;strong&gt;Inmunostaining&lt;/strong&gt;</td></tr><tr><td>Phosphate buffer saline (-MgCl&lt;sub&gt;2&lt;/sub&gt;, -CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Gibco</td><td>10010031</td><td>Store at room temperature</td></tr><tr><td>PBST, PBS made up with 0.1% TWEEN 20&amp;nbsp;</td><td>&amp;nbsp; Scientific Laboratory Supplies Ltd</td><td>EC607&amp;nbsp;</td><td></td></tr><tr><td>Methanol</td><td>&amp;nbsp; 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 colspan="4">&lt;strong&gt;Cell culture and Functional assay&lt;/strong&gt;</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>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/12604013&quot;&gt;12604013&lt;/a&gt;</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）。...

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

Universidad San Sebastian

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

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

界定和由人多能干细胞的肝细胞样细胞的可扩展代

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

科研

发育生物学

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

人诱导多潜能干细胞衍生肝细胞的药物发现方法

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Current differentiation procedures generate human hepatocyte-like cells (HLCs) displaying a mixture of fetal and adult traits. To improve cell phenotype, we have fully defined our differentiation procedure and the cell niche, resulting in the generation of cell populations which display improved gene expression and&#160;function. While these studies mark progress, the ability to generate large quantities of multi well plates for screening has been limited by labour intensive procedures and batch to batch variation. To tackle this issue, we have developed a semi-automated platform to differentiate pluripotent stem cells into HLCs. Stem cell seeding and differentiation were performed using liquid handling and automatic pipetting systems in&#160;96-well plate format. Following the differentiation, cell phenotype was analyzed using automated microscopy and a multi well luminometer.

This protocol describes a semi-automated approach to produce hepatocyte-like cells from human pluripotent stem cells in a 96 well plate format. This process is rapid and cost-effective, allowing the production of quality assured batches of hepatocyte-like cells for basic and applied human research.

多能干细胞半自动化生产肝样细胞的研究

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic ...

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

从多能干细胞中无血清自由生产三维人类肝球

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

肝细胞样细胞从人类胚胎干细胞群的鲁棒代

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

生物学

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

协议主要人肝细胞的分离和相应的非实质肝细胞的主要种群

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

小鼠肝部分切除术后再生肝细胞的分离

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

医学

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to shortages in donor organ availability. Organ scarcity also affects the routine supply of human hepatocytes for basic research and the clinic. Therefore, the development of renewable sources of human liver progenitor cells is desirable and is the goal of this study. To be able to effectively generate and deploy human liver progenitors on a large scale, a reproducible hepatic progenitor differentiation system was developed. This protocol aids experimental reproducibility between users in a range of cell cultureware formats and permits differentiations using both, human embryonic and induced pluripotent stem cell lines. These are important advantages over current differentiation systems that will enhance the basic research and may pave the way towards clinical product development.

The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

使用定义的分化系统从多能干细胞的肝祖原体规范

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to ...

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.

小鼠肝细胞类器官的建立与遗传操作

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the ...

用于药物发现的生理相关全人 2D+ 肝脏系统

An All-Human Hepatic Culture System for Drug Development Applications

Finding a long-term, human-relevant culture model for primary human hepatocytes (PHHs) for pharmacological and toxicological studies remains a challenge. Current in vitro model platforms are often inconvenient and complex, lack phenotypic stability over time, and do not support multiple PHH lots, lacking experimental reproducibility and flexibility. Here, we provide a detailed protocol for the thawing, plating, and maintenance of an all-human 2D+ hepatic system (TV2D+), which takes advantage of standard two-dimensional (2D) culture techniques and equipment while maintaining the longevity and phenotypic stability over time that typically accompany more complex three-dimensional (3D) systems. The results show attachment and percent plateability in TV2D+ as a function of PHH seeding density, as well as stable functionality for at least 2 weeks in culture. A range of PHH seeding densities are assessed to achieve a successful long-term culture. When established properly, the PHHs in TV2D+ organize into hepatocyte colonies, express a hepatic-specific marker, and maintain viability, architectural integrity, and physiologically relevant levels of albumin and urea. This unique combination of attributes makes the TV2D+ system a suitable hepatic model for a variety of pharmacological and toxicological applications.

作者聚焦:为药理学和毒理学应用开发简单而强大的肝脏模型 

Recent technical advances enabled the scaled production of an in vitro platform for drug metabolism and toxicity applications. An all-human 2D+ hepatic system (TV2D+) provides physiologically relevant results using traditional two-dimensional culture methods. This protocol will support end-users in the setup, maintenance, and application of the system.

用于药物开发应用的全人肝培养系统

Finding a long-term, human-relevant culture model for primary human hepatocytes (PHHs) for pharmacological and toxicological studies remains a challenge. ...