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

DCH、MBとFKは基金に関するEPSRCフォローによってサポートされていました。 BL-VとDSがそれぞれのMRC博士studentshipsによってサポートされていました。 KCは、英国再生医療プラットフォームからの資金によってサポートされていました。

PHNGAD（ポリ[1,6-hexanodiol /ネオペンチルグリコール/ジ（エチレングリコール）-alt - アジピン酸]ジオール）の合成<img alt="スキーム1" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme1.jpg" width="500" /> スキーム1：PHNAGDの合成 PHNAGDの合成の模式図。。 PHNAGDは、1,6 - Hexanodiol、ジエチレングリコール、neoppentylグリコールとアジピン酸との反応により調製した。 PHNAGD、ポリ[1,6-hexanodiol /ネオペンチルグリコール/ジ（エチレングリコール）-alt - アジピン酸]ジオール。 <ol><li>残留水を除去し、真空オーブン中で48時間40℃でのモノマー1,6 - ヘキサンジオール、ジ（エチレングリコール）及びネオペンチルグリコールに熱処理を適用する。真空下で室温まで寒さにできるようにします。 </li><li>二口丸底フラスコに攪拌棒を備えたディーン·スターク装置に接続する各モノマーの22ミリモルを加え、アジピン酸（55ミリモル）を加えた。 </li><li>真空下でアセンブリ全体を置き、静かにGLAを加熱フラスコ中の化学物質の添加中の任意の吸湿を避けるために、6時間40℃でssware。 </li><li> 、一滴ずつ、シリンジを介して追加の触媒チタン（IV）ブトキシドの0.0055モル。 </li><li> 24時間N 2雰囲気下、180℃で反応混合物を撹拌し、そしてディーン-スタークトラップに残留水を集める。製品が室温まで冷却させる。 </li></ol> PU134の2の合成<img alt="スキーム2" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme2.jpg" width="500" /> スキーム2：PU134の合成 PU134は、4,4 &#39; -メチレンビス（フェニルイソシアネート）の2.0当量とPHNGADの1.0当量との反応により調製されたポリウレタン134の合成の略図、1.0を添加した。 1,4 - ブタンジオール連鎖延長剤の当量。 <ol><li> 4の2当量とポリオールPHNGAD 1当量の量（Mn〜1,800ダルトン、3.2ミリモル）を混ぜ無水N 4 &#39; -メチレンビス（フェニルイソシアネート）（6.4ミリモル）、Nジメチルホルムアミド（12 ml）を加えた。 </li><li> N 2雰囲気下、70℃で反応混合物を、攪拌する。 </li><li>触媒チタン（IV）ブトキシド（0.8％重量）を一滴ずつ、シリンジを介して追加します。 </li><li> 1時間後、鎖延長剤1,4 - ブタンジオール（3.2ミリモル）の1当量を加える。 90℃に温度を上昇させ、N 2雰囲気下で24時間撹拌した。 </li><li>反応後、沈殿が生じるまで反応溶液に滴下し（ヘキサンまたは水を使用することもできる）のジエチルエーテルを加えて沈殿させることによりポリウレタンを収集する。 </li><li> 5分間5,300×gで解決策を遠心。 </li><li>上清を除去し、溶媒が蒸発するまで、真空オーブンで40℃で乾かす。 注するために、反応の最終生成物は、分子量分布に関する適切なパラメータを有することを確実にするために、機能的grouポリマー、溶融およびガラス転移温度のpsが、さまざまな分析技術および方法は、ゲル浸透クロマトグラフィーまたはFITR分光法を用いることができる。 </li></ol> PU134ソリューションの調製<ol><li>ガラス瓶にPU134を200mg秤量する。 </li><li>クロロホルム、1クロロホルムとトルエンの組み合わせ：1の比率、テトラヒドロフラン、及び1テトラヒドロフラン及びdicloromethaneの組み合わせ：1の比の溶媒の数が2％の最終濃度にPU134を希釈する。 注：右の溶媒の選挙は、使用するポリマーに応じて変化する。異なる溶媒は、ポリマーの可溶化に影響を与えることができる異なる沸点を有する。 </li><li>溶液が均一になり、沈殿物が観察されなくなるまで、200 MOT /分の速度でシェーカーを用いて室温で20分間溶液を激しく振る。 </li></ol> PU134とスライドガラスの4。塗装<ol><li> ROUを配置スピンコーター上のND 15ミリメートル2カバースリップ。 </li><li>ピペットを用いて各PU134溶液50μlを適用します。比に比例表面ボリュームを保つ必要なカバーガラスサイズに応じてPU134溶液の量を調整します。 </li><li> 23 x gで7秒間、各カバースリップスピン。 </li><li>滅菌前に少なくとも24時間、室温で空気乾燥カバースリップ。 </li></ol>カバースリップの5。照射<ol><li> 12分間実験室の照射装置を用いて10グレイの線量を適用することにより、ポリマーで被覆されたカバースリップをガンマ - 照射する。 </li><li> UV-16分間、30 W、UV電球の各側面を用いてポリマーコーティングされたカバースリップを照射する。 </li><li>スライドのサイズに応じて適切な組織培養プレートにポリマー被覆カバースリップを置きます。 </li></ol> 6。走査型電子顕微鏡<ol><li>圧力5×10 -1ミリバールの雰囲気下で200秒間スパッタリングによる金コートポリマーカバースリップ。 </li><li>マイクログラムをキャプチャ二次電子撮像モードで20kVの加速電圧での走査型電子顕微鏡を用いてポリマーコーティングされたカバースリップのraphs。 </li></ol> 7。原子間力顕微鏡の観察<ol><li>ポリマー表面の20×20μmの領域をスキャンします。 </li><li> 1.32ヘルツから1.60 Hzのスキャンレートを設定します。 </li><li>スキャンされた領域内の512×512ピクセルの解像度を設定します。 </li><li>平均画像データプレーンから採取した高さ偏差の平均値を用いて、コーティングの平方根（RMSまたはさRq）の平均平方根を計算し、のように表さ。 <img fo:content-width="1in" src="/files/ftp_upload/51723/51723eq1.jpg" width="123" /> Ziが現在のZ値であり、Nは、所定の領域内の点の数である。 </li><li> 7.5偏差を計算し、または使用して、画像の表面粗さ（Ra）を意味し、 <img fo:content-width="1.5in" src="/files/ftp_upload/51723/51723eq2.jpg" width="154" /> Z（x）はtで記述する関数である。彼は、高さ（Z）と評価長さ &quot;L&quot;を超える試料の位置（x）に関して分析プロファイル表面。 Raは中心面に対する表面の平均値を表す。 </li></ol> 8。細胞培養および分化<ol><li>文化·ヘイ17で説明したようにヒト胚性幹細胞株（ヒトES細胞）H9を区別。 </li><li>解離試薬を用いて細胞を外し、Szkolnicka 18,19で説明するように、無血清培地の存在下で、分化過程の9日目でPU134被覆スライドにドラッグreplate。 注：酵素的細胞解離の使用は物理的な細胞剥離に好適である。 </li></ol> 9チトクロームP450機能アッセイ<ol><li>製造者の指示に従ってCYP3A活性を測定する。 </li><li>細胞なしのhESCに由来する13日目または19日目での肝細胞、およびメディアをインキュベート - ネガティブコントロールとして、適切なSUBSTRで37℃で5時間食べた。 </li><li> 、細胞および培地からの上清を回収し、製造業者の指示に従ってアッセイを実施しています。 </li><li>表面積（cm 2）でのCYP活性と正規化のレベルを測定する。 </li></ol> 10免疫染色<ol><li> PBSで洗浄するのhESC派生肝細胞は、1分間、2回繰り返す。 </li><li> 10分間、-20℃での氷冷100％の細胞を固定するために、メタノール、場所を追加します。 </li><li> 5分間のPBSで細胞を洗浄し、二回繰り返します。 </li><li> RTで1時間PBS / T（0.1％トゥイーン）/ 10％BSAで細胞をインキュベートする。 </li><li> PBST液を吸引し、PBS / T（0.1％トゥイーン）/ 1％BSA中に希釈した適切な一次抗体を追加し、穏やかに撹拌し、O / N、4℃でインキュベートする。 </li><li> 5分間のPBS / T（0.1％トゥイーン）/ 1％BSAで細胞を洗浄し、三回繰り返して。 </li><li> ％のBSA / 1×PBS / T（0.1％トゥイーン）中で適切な抗体を希釈し、細胞に追加し、穏やかに撹拌しながら室温で1時間、暗所でインキュベートする。。 </li><li> 5分間のPBSで細胞を洗浄し、3回繰り返します。 </li><li>各ウェルに、気泡の数を減らすために穏やかにカバースリップを追加し（千DAPI 1を含む）MOWIOL 488を追加する。暗闇の中で4℃で保存固定した細胞。適切なフィルターと蛍光灯顕微鏡を用いて染色し観察します。最適化された一次および二次抗体は、 表1に記載されています。 </li></ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-+and+microscale+holes+modulate+cell-substrate+adhesion,+cytoskeletal+organization,+and+-beta1+integrin+localization+in+SV40+human+corneal+epithelial+cells.">Nano- and microscale holes modulate cell-substrate adhesion, cytoskeletal organization, and -beta1 integrin localization in SV40 human corneal epithelial cells.</a> IEEE Transactions on Nanobioscience. 5 (4), 273-280 (2006).</li><li>Hamilton, D. W., Brunette, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+substratum+topography+on+osteoblast+adhesion+mediated+signal+transduction+and+phosphorylation.">The effect of substratum topography on osteoblast adhesion mediated signal transduction and phosphorylation.</a> Biomaterials. 28 (1), 1806-1819 (2007).</li><li>Engler, A. 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>

DCHはCSO、FibromEdプロダクツ株式会社における取締役、創業者でFibromEd製品株式会社MBおよびJPIにおける株主である創業者の株主である

幹細胞から肝細胞を生成するために使用される現在の方法の多くは、動物由来の未定義のマトリックスに依存している。これらの基板は、アプリケーションに重大な障壁を表す、細胞の機能および安定性に影響を与える、費用がかかり、非常に可変であることができる。そこで、幹細胞由来肝細胞の培養を支持する合成材料のためのスクリーンを行った。私たちは、同定した、堅牢な肝細胞?...

生物学的材料は、広く、多能性幹細胞1の維持および分化に使用されている。可能にしながら、これらの生物学的基質は、多くの場合、未定義の構成要素の無数が含まれています。マトリゲルは、幹細胞の培養および分化のための一般的に使用される基板である。残念ながら、その可変組成は、細胞の機能および表現型に影響を与える。代替、複数の定義された生物学的マトリックスの多様2-7使用されてきたが、それらの動物由来または貧弱な拡張性は、彼らに工業的な製造には適さない候補にする。したがって、定義された組成と信頼できる性能を有する合成代替物の同定は、幹細胞研究の重要な目標である。 未定義の細胞培養基質の制限を克服する試みにおいて、化学と生物学との間の学際的コラボレーションは、細胞表現型をサポートする能力を有する合成材料を同定した。シンセエティックな基板は、スケーラブルであり、費用対効果、および複雑な3次元構造に製造することができ、 生体内環境を模倣する。によるこれらの特性に合成基質は、広く多くの細胞型8-10の分化を支持し、駆動するために使用されてきた。 Advancedおよびハイスループットアッセイは、大規模なライブラリーから、合成材料の迅速なスクリーニングを容易にし、生物医学の研究開発11-13の幅広いアプリケーションとの柔軟な特性を有する新規材料を提供してきました。高いスループットを利用して、高分子マイクロアレイスクリーニング技術は、急速にヒト幹細胞由来の肝細胞の維持に適した単純なポリウレタン（PU134）を同定した。このポリマーは、肝細胞の分化および機能14-16に関して動物由来の基質よりも優れていることが判明した。私たちは、その後の効果をアクセスするための塗布条件、地形や滅菌プロセスを最適化した肝細胞機能と寿命を安定化するポリマーの性能に。これは、セルベースの​​モデリングと再生医療アプリケーションのための肝細胞生物学の基礎を理解することに関して重要な意味を持っている。 ここで説明する技術は、合成ポリマーの表面が細胞表現型を維持するために最適化することができる方法の例を示している。私たちは、効率的な無血清肝細胞分化プロトコルでこの技術の組み合わせは、 インビトロモデリングや再生医療で使用するための肝細胞のスケーラブルな生産を提供する可能性を持っていることを信じています。

<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.

ポリマー溶媒は、ポリマーコーティングされた表面の形状に影響を与える ポリウレタン134は、トルエンまたはテトラヒドロフランまたはジクロロメタンとの単独または組み合わせのいずれかで、クロロホルムに溶解し、ガラススライドは、異なる製剤でスピンコートした。走査電子顕微鏡（SEM）および原子間力顕微鏡（AFM）は、ポリマーコーティング（ <str...

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

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

この記事では、ヒト肝細胞を由来幹細胞の長期、安定的な培養のためにポリマーコーティングされた表面の開発に注力していきます。

最適化された合成表面を使用した肝細胞表現型の安定化

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 ビュー.  University of Edinburgh. この記事では、ヒト肝細胞を由来幹細胞の長期、安定的な培養のためにポリマーコーティングされた表面の開発に注力していきます。 

ビデオ: 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.

ヒト多能性幹細胞から肝細胞様細胞の定義され、スケーラブルな世代

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

発生生物学

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.

創薬における多能性幹細胞由来肝細胞様細胞を誘導人間を使用して

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.

多能性幹細胞からの細胞のような肝細胞の半自動化生産

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.

多能性幹細胞からの3次元ヒト肝球の血清自由産生

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.

ヒト胚性幹細胞集団から肝細胞様細胞の堅牢な生成

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.

初代ヒト肝細胞と非実質肝細胞の対応する主な集団の単離のためのプロトコル

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.

マウスにおける肝部分切除後の再生肝細胞の単離

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.

定義された分化系を用いた多能性幹細胞からの肝前駆体仕様

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.

マウス肝細胞オルガノイドの確立と遺伝子操作

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

創薬のための生理学的に関連性のある全ヒト2D+肝臓システム

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.

著者スポットライト:薬理学的および毒物学的アプリケーションのためのシンプルで堅牢な肝臓モデルの開発 

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.

創薬応用のための全ヒト肝培養システム

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