YSS, MB ve FK Fonuna ilişkin EPSRC Takip tarafından desteklenmiştir. BL-V ve DS her MRC Doktora Studentships tarafından desteklenmiştir. KC İngiltere Rejeneratif Tıp Platformu fon tarafından desteklenmiştir.

PHNGAD (poli [1,6-hexanodiol / neopentil glikol / di (etilen glikol) -Alt-adipik asit] dio) 1. sentezi <img alt="Şema 1" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme1.jpg" width="500" /> Şema 1: PHNAGD sentezi PHNAGD sentezinin şematik gösterimi.. PHNAGD 1,6-Hexanodiol reaksiyonu, dietilen glikol, neoppentyl glikol ve adipik asit ile hazırlandı. PHNAGD, poli [1,6-hexanodiol / neopentil glikol / di (etilen glikol) -Alt-adipik asit] dio. <ol><li> Herhangi bir artık suyun ayrılması için bir vakumlu fırın içinde 48 saat boyunca 40 ° C &#39;de monomerler 1,6-heksandiol, di (etilen glikol) ve neopentil glikol için ısıl işlem uygulanır. Vakum altında oda sıcaklığına soğumuş izin verin. </li><li> Bir Dean-Stark aparatı ile karıştırıldı çubuğu ile donatılmış ve bağlı olan, bir iki-boyunlu yuvarlak tabanlı bir şişeye, her monomer ve adipik asit (55 mmol), 22 mmol ekleyin. </li><li> Bir vakum altında bütün tertibatım yerleştirin, yavaşça GLA ısıtmakşişe içine kimyasal maddenin ilave edilmesi sırasında nem emilmesini önlemek için, 6 saat boyunca 40 ° C, ssware. </li><li> Damla katalizör titanyum (IV) butoksit 0.0055 mol damla şırınga ile ilave edin. </li><li> 24 saat boyunca, bir N2 atmosferi altında 180 ° C&#39;de, reaksiyon karışımı, karıştırın ve Dean-Stark tuzağı içinde kalan suyun toplanması. Ürün oda sıcaklığına soğumaya bırakın. </li></ol> PU134 2. sentezi <img alt="Şema 2" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme2.jpg" width="500" /> Şema 2: PU134 sentezi PU134 bir 4,4 &#39;-metilenbis (fenil izosiyanat) 2.0 eşdeğer bir PHNGAD 1.0 eşd reaksiyonu ile hazırlandı poliüretan 134. sentezinin şematik temsili, 1.0 ilave edildi. bir 1,4-bütandiol zincir uzatıcının eşdeğer. <ol><li> 4, iki eşdeğeri ile poliol PHNGAD bir eşdeğeri (Mn ~ 1800 Da, 3.2 mmol) karıştırınSusuz N &#39;4-metilenbis (fenil izosiyanat) (6.4 mmol) N, N-dimetilformamid (12 mi). </li><li> Bir N2 atmosferi altında 70 ° C de, reaksiyon karışımı, ilave edin. </li><li> , Şırınga ile, katalizör ekleme damla damla titanyum (IV) butoksit (0.8% wt). </li><li> 1 saat sonra, zincir uzatıcı 1,4-bütandiol (3.2 mmol) bir eşdeğeri ilave edin. 90 ° C de sıcaklığı artar ve bir N2 atmosferi altında 24 saat için karıştırın. </li><li> Reaksiyonu takiben, çökelti oluşacak kadar reaksiyon çözelti içine damla damla (de kullanılabilir heksan ya da su), dietil eter ilave edilerek çökeltme poliüretan toplar. </li><li> 5 dakika boyunca 5,300 x g&#39;de çözümü santrifüj. </li><li> Süpernatant süzün ve çözücü buharlaşana kadar bir vakum fırınında 40 ° C&#39;de kurutunuz. Not: amacıyla reaksiyonun nihai ürünün molekül ağırlığı dağılımına ilişkin parametreler doğru sahip olduğundan emin olmak için işlevsel Groupolimer ve ergitme ve cam geçiş sıcaklıklarının PS, çeşitli analitik teknikler ve yöntemler, jel geçirgenlik kromatografisi veya Ramazan spektroskopi gibi, kullanılabilir. </li></ol> PU134 Çözümleri 3. hazırlanması <ol><li> Bir cam şişe içine 200 mg PU134 tartılır. </li><li> Kloroform, 1 kloroform ve toluen bir kombinasyonu: a çözücüler sayısındaki% 2&#39;lik bir nihai konsantrasyona kadar seyreltilir PU134 1 oranında, tetrahidrofuran, ve kombinasyon 1 tetrahidrofuran ve diklorometan arasında: 1 oranında. Not: Sağ çözücünün seçimi, kullanımı polimere bağlı olarak değişir. Değişik çözücüler polimerin çözünürlüğünü etkiler farklı kaynama noktalarına sahiptir. </li><li> Çözelti homojen hale gelir ve çökelti gözleninceye kadar, 200 mot / dakika hızda bir çalkalayıcı kullanılarak, oda sıcaklığında kuvvetli bir şekilde 20 dakika süre ile çözelti çalkalayın. </li></ol> PU134 Cam Slaytlar 4. Kaplama <ol><li> Bir rou yerleştirinSpin lak nd 15 mm 2 lamel. </li><li> Bir pipet kullanılarak her PU134 solüsyonu 50 ul uygulayın. Oranı ile orantılı yüzeye hacim tutarak gerekli lamel boyutu için buna PU134 çözümün ses seviyesini ayarlayın. </li><li> 23 x g 7 saniye boyunca her lamel Spin. </li><li> Sterilizasyondan önce en az 24 saat boyunca oda sıcaklığında kuru hava lamel. </li></ol> Lamel 5. Işınlama <ol><li> 12 dakika süre ile, laboratuar ışınlama kullanılarak 10 Grays bir dozunun uygulanmasıyla polimer kaplı lamel Gama-ışın tedavisi. </li><li> 16 dakika her iki taraf için bir 30 W, UV ampulü kullanarak polimer kaplı lamel ışın UV. </li><li> Slayt boyutuna uygun olarak uygun bir doku kültürü plakasında, polimer kaplı lamel yerleştirin. </li></ol> 6. Taramalı Elektron Mikroskobu <ol><li> Basınç 5 x 10 milibar -1 atmosferinde 200 saniye boyunca püskürtme ile altın kaplama polimer lamel. </li><li> Microg yakalayınİkincil elektron görüntüleme modunda 20 kV bir hızlandırma voltajı bir tarama elektron mikroskobu kullanılarak polimer ile kaplanmış lamel raphs. </li></ol> 7. Atomik Kuvvet Mikroskobu Gözlemler <ol><li> Polimer yüzeyin 20 x 20 um tarama alanı içinde, bir. </li><li> 1.32 Hz 1.60 Hz tarama hızı ayarlayın. </li><li> Taranan bölgede 512 x 512 piksel çözünürlüğe ayarlayın. </li><li> Kök ortalama resim veri düzleminden alınan yükseklik sapmalarının ortalaması kullanılarak kaplamanın karesi (RMS veya Rq) hesaplayın ortalama olarak ifade edilen: <img fo:content-width="1in" src="/files/ftp_upload/51723/51723eq1.jpg" width="123" /> Nerede Zi akım Z değeri ve N belirli bir alanda nokta sayısıdır. </li><li> Kullanarak görüntünün 7,5 sapmasını hesaplayın ve ortalama yüzey pürüzlülüğüne (Ra), <img fo:content-width="1.5in" src="/files/ftp_upload/51723/51723eq2.jpg" width="154" /> Burada Z (X) t tarif fonksiyonuduro yükseklikte (Z) ve değerlendirme uzunluğu &quot;L&quot; üzerinden örnek konumuna (x) cinsinden analiz yüzey profili. Ra orta düzlemine yüzey nisbetle ortalama değeri temsil eder. </li></ol> 8. Hücre Kültürü ve Farklılaşma <ol><li> Kültür ve Hay 17&#39;de tarif edildiği gibi, insan embriyonik kök hücre çizgisi (HESC) H9 ayırt eder. </li><li> Bir ayırma reaktifi kullanılarak hücreleri ayırmak ve Szkolnicka 18,19 &#39;de anlatıldığı gibi bir serumsuz ortam varlığında, farklılaşma gün, 9 ile kaplanmış dilimlerin üzerine PU134 bunları Replate. Not: Enzimatik Çözülme fiziksel hücre ayrılması için tercih edilir. </li></ol> 9. Sitokrom P450 üzerinde fonksiyonel denemeler <ol><li> Üreticinin talimatlarına CYP3 aktivitesini ölçmek. </li><li> Hücreler olmaksızın HESC türetilmiş Gün 13 ve Gün 19 hepatositleri ve ortam inkübe - negatif kontrol olarak, uygun olan substr37 ° C&#39;de 5 saat boyunca yedi. </li><li> , Hücreler ve ortamdan süpernatantlar toplamak üreticinin talimatlarına göre analizini gerçekleştirmek. </li><li> Yüzey alanı (cm2) için CYP aktivite ve normalize seviyesini ölçer. </li></ol> 10. immün <ol><li> Yıkama HESC 1 dakika boyunca PBS ile hepatositleri türetilmiş, iki kez tekrarlanır. </li><li> 10 dakika boyunca -20 ° C&#39;de buz soğukluğunda% 100 hücreleri düzeltmek için, metanol, yer ekleyin. </li><li> 5 dakika boyunca PBS ile yıkayın ve hücreler iki kez tekrarlayın. </li><li> PBS / T (% 0.1 Tween), oda sıcaklığında 1 saat boyunca /% 10 BSA ile hücreleri inkübe edin. </li><li> PBST solüsyonu aspire ve PBS / T (% 0.1 Tween) /% 1 BSA içinde seyreltilmiştir ve uygun bir primer antikor ekleyin, hafif çalkalama O / N 4 ° C&#39;de inkübe edin. </li><li> 5 dakika boyunca PBS / T (% 0.1 Tween) /% 1 BSA hücreleri yıkayın ve üç kez tekrarlayın. </li><li> PBS / T (% 0.1 Tween) /% 1 BSA içinde, uygun bir antikor seyreltik hücrelere eklenir ve hafif çalkalanarak 1 saat boyunca oda sıcaklığında karanlıkta inkübe. </li><li> 5 dakika boyunca PBS ile hücreleri yıkayın ve üç kez tekrarlayın. </li><li> Her bir oyuğa hava kabarcıkları sayısını azaltmak için hafifçe bir lamel ekleyin: (1000 DAPI içeren 1) Mowiol 488 ekleyin. Mağaza karanlıkta 4 ° C&#39;de duran hücrelerin. Uygun bir filtre ve floresan lambası ile bir mikroskop kullanılarak boyama dikkate alınmalıdır. Optimize edilmiş birincil ve ikincil antikorlar, Tablo 1 &#39;de listelenmiştir. </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>

YSS STK, FibromEd Ürünler Ltd Direktörü, kurucusu ve FibromEd Ürünleri Ltd MB ve JPI bir hissedar olan kurucu ortaklar olduğunu

Kök hücrelerden hepatositleri üretmek için kullanılan mevcut yöntemlerin çoğu hayvan menşeli tanımlanmamış matrisler dayanır. Bu alt-tabakalar uygulama için önemli bir engel temsil eden hücre fonksiyonu ve stabilitesini etkileyen, pahalı ve son derece değişken olabilir. Bu nedenle, kök hücre elde edilen hepatositlerin kültürünü destekleyen sentetik malzemeler için bir ekran ısırttı. Biz belirledik, sağlam bir hepatosit farklılaşma yaklaşımı ile kombinasyon halinde PHNGAD, MDI ve bir ge...

Biyolojik maddeler, yaygın olarak, pluripotent kök hücreleri 1 bakım ve farklılaşması kullanılmıştır. Sağlarken, bu biyolojik substratların tanımlanmamış bileşenlerin sayısız içerir. Matrigel kök hücre kültürü ve farklılaşması için yaygın olarak kullanılan bir alt-tabakadır. Ne yazık ki, değişken bileşim hücre fonksiyonu fenotipi etkiler. Alternatif, daha tanımlı biyolojik matris çeşitli 2-7 kullanılıyor olsa, onların hayvansal kökenli veya kötü ölçeklenebilirlik onlara endüstriyel üretimi için uygun bir aday haline getiriyor. Bu nedenle, kök hücre araştırmalarında önemli hedefler tanımlanır kompozisyon ve güvenilir performansı ile sentetik alternatifleri, kimlik vardır. Tanımsız hücre kültürü yüzeylerin sınırlamaları aşmak için bir girişim, kimya ve biyoloji arasında disiplinler arası işbirliği hücre fenotipi desteklemek için kapasite ile sentetik malzemeler tespit ettik. Synthetik yüzeyler, etkili ölçeklenebilir maliyet ve in vivo ortamda taklit, karmaşık 3D yapılarına imal edilebilir. Bu özelliklere bağlı olarak, sentetik alt-tabakalar, çoğu hücre tipinde yaygın olarak 8-10 farklılaşmasını desteklemek ve sürmek için kullanılmıştır. Gelişmiş ve yüksek kapasiteli testler büyük kütüphanelerinden, sentetik malzemelerin hızlı tarama kolaylaştırdı ve biyomedikal araştırma ve geliştirme 11-13 geniş uygulamaları ile esnek özelliklere sahip yeni malzemeler teslim ettik. Yüksek verim, polimer mikro-dizi tarama teknolojisini kullanarak, hızla insan kök hücresi elde edilen hepatositlerin bakımı için uygun olan basit bir poliüretan (PU134), tespit. Bu polimer, hepatosit farklılaşımı ve fonksiyon 14-16 ile ilgili olarak, hayvan kaynaklı alt tabakalara üstün olduğu bulunmuştur. Biz sonradan etkilerini erişmek için kaplama koşulları, topografya ve sterilizasyon işlemini optimizehepatosit fonksiyonları ve ömrünü stabilize polimer performansı. Bu hücre tabanlı modelleme ve rejeneratif tıp uygulamalarında hepatosit biyolojinin temel anlayış açısından önemli etkileri vardır. Burada anlatılan teknoloji sentetik bir polimerin yüzey hücre fenotipi korumak için optimize edilebilir nasıl bir örneğini temsil eder. Verimli bir serumsuz hepatosit farklılaşma protokol ile bu teknolojinin kombinasyonu in vitro olarak modelleme ve rejeneratif tıpta kullanım için hepatosit ölçeklenebilir üretim sağlamak için bir potansiyele sahip olduğuna inanıyoruz.

<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.

Polimer çözücü polimer kaplanmış bir yüzey topografisi etkiler Poliüretan 134 tek başına ya da toluen ya da tetrahidrofuran ya da diklorometan ve cam slaytlar farklı formülasyonlar ile kaplı dönüşü ile kombinasyon halinde, kloroform içinde çözüldü. Tarama elektron mikroskopi (SEM) ve atomik kuvvet mikroskopisi (AFM) (Şekil 1) polimer kaplamaların fiziksel özelliklerini karakterize etmek için kullanıldı. Toluen veya kloroform kulla...

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

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Bu makalede, uzun vadede polimer kaplı yüzeylere geliştirilmesi üzerinde durulacak, kök hücre istikrarlı kültür, insan hepatositleri türetilmiş.

Optimize Sentetik Yüzeyler Hepatosellüler Fenotip kullanma Stabilize

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

Research

JoVE Journal

Chemistry

10.2K Görüntüleme.  University of Edinburgh.  Bu makalede, uzun vadede polimer kaplı yüzeylere geliştirilmesi üzerinde durulacak, kök hücre istikrarlı kültür, insan hepatositleri türetilmiş. 

Video: 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.

Tanımlı ve hepatosit benzeri Hücrelerinin Ölçeklenebilir Nesil insan pluripotent kök hücreleri gelen

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

Gelişim Biyolojisi

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.

İnsan kullanarak Pluripotent kök hücre kaynaklı hepatosit benzeri hücreler indüklenen ilaç bulma için

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.

Hepatosit Pluripotent kök hücreler hücreleri gibi yarı otomatik üretimi

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.

Pluripotent kök hücrelerinden üç boyutlu ınsan Hepatokürlerin serum ücretsiz üretimi

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.

İnsan Embriyonik Kök Hücre Popülasyonlar Hepatosit gibi Hücreleri Sağlam Nesil

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

Biyoloji

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

Karaciğer Hastalığı Çalışmaları için Periferik Kandan İnsan Karaciğer Sferoidleri

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

Biyomühendislik

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.

İlköğretim İnsan Hepatositlerin İzolasyonu ve Non-parankimal karaciğer hücrelerinin Binbaşı popülasyonları İlgili Protokolü

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.

Farelerde Parsiyel Hepatektomi Sonrası Rejenere Hepatositlerin İzolasyonu

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

Tıp

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.

Tanımlanmış Bir Farklılaşma Sistemi Kullanarak Pluripotent Kök Hücrelerden Hepatik Progenitör Spesifikasyonu

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.