The authors thank the support of the Zell Family Foundation. We recognize support from the Northwestern Memorial Foundation Dixon Translational Research Grants Initiative, the American Society of Transplant Surgeon&#8217;s Faculty Development Grant, and a Research Grant for the Young Investigator from the National Kidney Foundation of Illinois. We acknowledge support from the Robert R. McCormick Foundation. This work was also supported by NIDDK K08 DK10175 to J.A.W. Imaging and histology cores used for this research is supported by the Mouse Histology and Phenotyping Laboratory, Electron Probe Instrumentation Center (EPIC), and Simpson Querrey Institute Equipment Core at Northwestern University, and a Cancer Center Support Grant (NCI CA060553). The authors would like to acknowledge the Northwestern University Microsurgery Core for rodent kidney procurements. Evaluation of renal tubular epithelia morphology following recellularization conducted in the Fluorescence Microscopy Shared Resource supported by P30 CA118100.

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+methods+of+porcine+kidneys+for+whole+organ+engineering+using+a+high-throughput+system.">Decellularization methods of porcine kidneys for whole organ engineering using a high-throughput system.</a> Biomaterials. 33, 7756-7764 (2012).</li><li>Vishwakarma, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+natural+three-dimensional+goat+kidney+scaffold+for+the+development+of+bioartificial+organ.">Preparation of natural three-dimensional goat kidney scaffold for the development of bioartificial organ.</a> Indian journal of nephrology. 24, 372-375 (2014).</li><li>Ross, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+proliferate+and+differentiate+when+seeded+into+kidney+scaffolds.">Embryonic stem cells proliferate and differentiate when seeded into kidney scaffolds.</a> J Am Soc Nephrol. 20, 2338-2347 (2009).</li><li>Song, J. 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The authors have nothing to disclose.

記載の脱細胞化プロトコルは、一貫して血管内皮細胞7,17に加えて、（近位および遠位尿細管の両方由来の）ヒト腎皮質尿細管上皮細胞の培養のための3次元テンプレートとして機能する完全無細胞腎ECMを生成します。カニューレを挿入し、腎血管系は、このように灌流脱細胞化、細胞播種、長期灌流培養、およびレサズリン灌流プロトコルを有効にする、バイオリアクターのセットア...

An erratum was issued for <a href="http://www.jove.com/video/53271/epithelial-cell-repopulation-preparation-rodent-extracellular-matrix">Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development</a>.
The human RCTEC/RCTE cell stocks used here and in Caralt et al., 2015, Uzarski et al., 2015 were originally provided by Dr. Loghman-Adham (then at St. Louis University) to co-author Dr. Wandinger-Ness (University of New Mexico) through an MTA in 2001. The SV40 immortalized human RCTEC/RCTE cells were characterized as being of distal tubule cell line as detailed in Loghman-Adham et al., 2003. On the basis of recent short tandem repeat (STR) DNA sequencing (Performed by IDEXX BioResearch) of the earliest passages of the RCTEC/RCTE cell stocks it became evident the cells were of mixed lineage. Further analyses of PCR products using QIAxcel capillary electrophoresis demonstrated the presence of a canine product. The product was sequenced and established to be of canine origin. A set of canine specific STR markers were compared to the sample and showed that the sample had a genetic profile with 92% identity to the MDCK cell line.&#160; Later cell stocks that were used in the present publication were STR profiled and showed drift to 100% MDCK lineage. Despite being of canine origin, rather than human as was previously thought, MDCK is similarly a distal tubule epithelial cell line. For this reason, previous interpretation and conclusions drawn using these cells here and in Caralt et al., 2015, Uzarski et al., 2015 remain sound, but for purposes of rigor, reproducibility and experimental validation by others we report on this misidentification. This information will also be reported for listing on the International Cell Line Authentication Committee (ICLAC) database (http://iclac.org).

Erratum: Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

末期腎不全に罹患している患者の数が増加し続けるように、移植のために利用可能なドナーの腎臓の多数の深刻な、成長不足しています。腎臓移植を待つ-リストされた候補者の継続的上昇数は需要1,2にカスタマイズされた、移植可能な腎臓移植の開発を究極の目標と腎臓臓器工学の研究を促してきた。の需要を満たすことができないこと患者自身の細胞から機能する腎臓組織を構築することは、生涯免疫抑制の必要性を排除する患者が腎臓移植を待っている透析に費やす時間の量を減少させ、慢性腎疾患を有するより多くの患者に救命移植を延長します。 患者由来の細胞を用いて、腎臓組織を生物工学に向けた最初のステップは、腎実質のための支援基質となる足場を開発することである（ 例えば、管状epitheLIAL）、間質線維芽細胞、および血管細胞増殖。自然の臓器の細胞外マトリックス（ECMの）由来の生体材料足場は、それらの天然の生物学的組成物を含む、組織工学での使用に望ましいものにいくつかの特徴を持っています。生理的機能を付与するための適切なマクロとミクロ構造;および細胞の生体適合性、細胞接着、遊走、および3を改造建設組織を促進します。腎組織再生のための足場を製造するための有望な方法は、腎ECM 4の複雑な天然のタンパク質組成物の多くを保存する臓器の固有のアーキテクチャの複雑さを保持し、ボトムに伴う困難を克服する同種異系または異種腎臓の脱細胞化によるものです足場再細胞化5の後に発展する細胞への血管供給を提供することによって、厚い細胞化組織のエンジニアリングまで。 灌流脱細胞化は、プロセスでありますこれは界面活性剤、酵素、または他の細胞破壊するソリューションを均一臓器6の血管網を介して配信されます。この戦略は破棄ヒト腎臓9から無細胞腎テンプレートの開発によって証明されるように、全体の臓器工学6-8のための生物学的テンプレートが、3次元（3D）のように無細胞器官系ECM足場を導出するために効率的な方法として確立されています大動物（ 例えば、ブタ 10、ヤギ11）および齧歯類源12から得られた異種腎臓。特に、げっ歯類などの小型動物モデルの使用は、より少数の細胞および幹細胞由来の組織と同様に、細胞数は、通常、限定された臓器の再細胞化の研究のために特に有用である培養培地を必要とします。説明脱細胞化プロトコルの目的は、腎臓の再生のための三次元足場システムとして使用することができる無細胞腎ECMを生成することであるstructurヒト腎臓皮質細管上皮（RCTE）細胞と、本実施例では再増殖しているネフロン細管を含め、ES。我々は以前、以前に報告されている他の方法よりも迅速で最適な、界面活性剤ベースのラット腎臓脱細胞化プロトコル7、（約1日）の当社の厳格な評価（ロスら .- 5日12、Song ら .- 4.5日を記載しました13）、前の報告12~15より脱細胞化中に変性剤ドデシル硫酸ナトリウム（SDS）のかなり低い濃度（0.1％）の臓器を露出させます。 研究の限られた数は、（他の場所で1レビュー）細胞の再増殖12-16ための3D足場として脱細胞化およびその後の使用のためにげっ歯類の腎臓の使用を記載しています。このプロトコルでは、無細胞腎臓足場を製造するために私たちの前に確立、最適な脱細胞化戦略の詳細な説明を提供しますスプラーグドーリーラットの腎臓7から。デュアル播種とメンテナンス灌流培養17が可能なカスタムデザインの灌流バイオリアクターを使用して、我々は一貫して、これらの脱細胞化マトリックス中の管状部品を再増殖増殖し、一週間以上のための灌流培養で生存したヒトRCTE細胞と無細胞腎臓足場を再細胞化。 17研究以前に細胞毒性のために使用される安価な、非細胞傷害性、および非侵襲性の代謝性評価- -時間7にわたって再細胞化腎臓内の細胞の生存度および増殖の指標を提供するために、我々はさらにレサズリン灌流アッセイの私たちの使用を示します。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TRITON X-100, Proteomics Grade, AMRESCO</td>
			<td>VWR</td>
			<td>M143-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate solution, BioUltra, for molecular biology, 20% in H2O</td>
			<td>Sigma-Aldrich</td>
			<td>05030</td>
			<td></td>
		</tr>
		<tr>
			<td>Peracetic acid solution, purum, ~39% in acetic acid (RT)</td>
			<td>Sigma-Aldrich</td>
			<td>77240</td>
			<td>Peracetic is flammable and corrosive. Prepare within a fume hood using appropriate personal protective equipment (e.g. gloves, goggles).</td>
		</tr>
		<tr>
			<td>200 proof ethanol</td>
			<td>VWR</td>
			<td>V1001TP</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigmacote, siliconizing reagent for glass and other surfaces</td>
			<td>Sigma-Aldrich</td>
			<td>SL2</td>
			<td>For treatment of bioreactor reservoirs. Referred to in text as siliconizing reagent.</td>
		</tr>
		<tr>
			<td>DMEM/F12 media</td>
			<td>Life Technologies</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning cellgro Fetal Bovine Serum Premium, Mediatech</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution, 100X 10,000 I.U. Penicillin 10,000 &#181;g/mL Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express (1X), Phenol Red</td>
			<td>Life Technologies</td>
			<td>12605-028</td>
			<td>Referred to in text as cell dissociating enzyme.</td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>R7017</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex L/S Digital Drive, 600 RPM, 115/230 VAC</td>
			<td>Cole-Parmer</td>
			<td>EW-07522-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex L/S large cartridges for 07519-05 and -06 pump heads.</td>
			<td>Cole-Parmer</td>
			<td>EW-07519-70</td>
			<td>Referred to in text as large pump cartridge.</td>
		</tr>
		<tr>
			<td>Masterflex L/S 8-channel, 4-roller cartridge pump head.</td>
			<td>Cole-Parmer</td>
			<td>EW-07519-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight 6&#34; specimen forceps, serrated</td>
			<td>VWR</td>
			<td>82027-438</td>
			<td></td>
		</tr>
		<tr>
			<td>*Kidney perfusion bioreactor</td>
			<td>WilMad Labglass</td>
			<td>*Custom designs</td>
			<td>Bioreactors are produced as described by WilMad Labglass. The designs have been described in depth in a previous publication.</td>
		</tr>
		<tr>
			<td>Perfusion Circuit Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24 G x 0.75 in. BD Insyte Autoguard shielded IV catheter (0.7 mm x 19 mm) made of BD Vialon biomaterial. Has notched needle. (50/sp, 200/ca)</td>
			<td>BD Biosciences</td>
			<td>381412</td>
			<td>Referred to in text as 24 gauge catheter.</td>
		</tr>
		<tr>
			<td>Masterflex PharMed BPT Tubing, L/S #14, 25&#39;</td>
			<td>Cole-Parmer</td>
			<td>HV-06508-14</td>
			<td>Referred to in text as peristaltic pump tubing.</td>
		</tr>
		<tr>
			<td>Peroxide-Cured Silicone Tubing, 1/16&#34;id X 1/8&#34;OD, 25 Ft/pack</td>
			<td>Cole-Parmer</td>
			<td>HV-06411-62</td>
			<td>Referred to in text as 1/16&#34; ID silicone rubber tubing.</td>
		</tr>
		<tr>
			<td>Masterflex platinum-cured silicone tubing, L/S 14, 25 ft.</td>
			<td>Cole-Parmer</td>
			<td>HV-96410-14</td>
			<td>Referred to in text as thick-walled 1/16&#34; ID silicone rubber tubing.</td>
		</tr>
		<tr>
			<td>VWR Silicone Tubing, 1/4&#34; ID x 0.5&#34; OD</td>
			<td>VWR</td>
			<td>89068-484</td>
			<td></td>
		</tr>
		<tr>
			<td>Acro 50 Vent Filters, Pall Life Sciences</td>
			<td>VWR</td>
			<td>28143-558</td>
			<td>Referred to in text as 0.2 micron vent filter.</td>
		</tr>
		<tr>
			<td>Cole-Parmer Luer Adapters, Male Luer Lock to 1/8&#34; ID, Nylon, 25/Pk</td>
			<td>Cole-Parmer</td>
			<td>T-45505-11</td>
			<td>Referred to in text as male Luer lock to 1/8&#34; barbed adapter.</td>
		</tr>
		<tr>
			<td>Cole-Parmer Luer Accessory, Male Luer Plug, Nylon, 25/Pk</td>
			<td>Cole-Parmer</td>
			<td>EW-45505-58</td>
			<td></td>
		</tr>
		<tr>
			<td>Female luer x female luer adapter, Nylon, 25/pk</td>
			<td>Cole-Parmer</td>
			<td>EW-45502-22</td>
			<td>Referred to in text as female Luer x female Luer adapter.</td>
		</tr>
		<tr>
			<td>Cole-Parmer Luer Accessory, Female Luer Cap, Nylon, 25/Pk</td>
			<td>Cole-Parmer</td>
			<td>EW-45502-28</td>
			<td></td>
		</tr>
		<tr>
			<td>Smiths Medical Large Bore Hi-Flo Stopcocks # MX4311L - 3-Way Hi-Flo Stopcock with Extended Male Luer Lock, Non-DEHP Formulation, Latex Free (LF), Lipid Resistant, Non-PVD, 50/cs</td>
			<td>Careforde Healthcare</td>
			<td>10254821</td>
			<td>Smiths Medical (vendor) catalogue number is #MX4311L.</td>
		</tr>
		<tr>
			<td>Other Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL BD Luer-Lok disposable syringe with BD Luer-Lok&#8482; tip.</td>
			<td>BD Biosciences</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm x 10 mm Easy Grip Culture Dish</td>
			<td>BD Biosciences</td>
			<td>353001</td>
			<td>Used to draw cell suspension into syringe for cell seeding.</td>
		</tr>
	</tbody>
</table>

epithelial cell repopulation and preparation of rodent extracellular matrix scaffolds for renal tissue development

このプロトコルは、無細胞の生成を詳述、まだ臓器規模な組織開発のための小規模なモデル基質として有用である生体機能、腎細胞外マトリックス（ECM）足場。スプラーグドーリーラットの腎臓がインタクト無傷、全腎臓足場を導出するために26時間かけて腎動脈にカテーテルを挿入することによりカニューレを挿入し、低濃度の界面活性剤（トリトンX-100、ドデシル硫酸ナトリウム（SDS））のシリーズで灌流されています灌流血管系、糸球体および尿細管。脱細胞化の後、腎足場は、カスタム設計された灌流バイオリアクター容器の内部に配置され、カテーテルは、腎動脈からなる灌流回路に接続されている：蠕動ポンプ。チューブ;そしてpH、溶存酸素、および圧力のためのオプションのプローブ。過酢酸、エタノール、及びpH（7.4）で平衡と足場を滅菌した後、腎臓足場はラーグ内の培地の灌流を介して、播種のために準備されます電子容量、37℃インキュベーターで維持し、5％CO 2。四十万人の腎皮質尿細管上皮（RCTE）細胞は、腎動脈を介して注入し、急速に生理的なレートに流れを減少させる前に、15分間、高流量（25 ml /分）と圧力（〜230 mmHgの）下足場に灌流されています（4 ml /分）。 RCTE細胞は、主に、腎皮質内の管状のECMニッチを移入増殖し、灌流培養の7日間で尿細管上皮構造を形成します。培養液中の44μMレサズリン溶液は細管形成の間の細胞の生存率および増殖の蛍光、レドックス系の代謝評価を提供するために、培地交換の際に1時間の腎臓を介して灌流されます。腎臓灌流バイオリアクターは、生化学的評価のための媒体の非侵襲的なサンプリングを可能にし、複数の入口ポートは、腎静脈または尿管を介して代替の逆行性播種を可能にします。これらのプロトコルは、腎臓での足場を再細胞化するために使用することができこのシステム内の迅速な評価のための血管内皮、尿細管上皮および間質線維芽細胞を含む、様々な細胞型、。

腎臓は、順次、水 ​​で灌流し、以前に確立された、最適な脱細胞化プロトコル（ 図1A、Bを参照してください）7、26時間かけて徐々に透明になるに従って洗剤溶液（1％トリトンX-100、0.1％SDS）を希釈し、として図2Aに示します。得られた無細胞腎臓足場は、細胞を欠いていると灌流プロトコルに従って破損していない無傷の腎被膜、でサポートされている粘...

Watch this Scientific Journal Video about Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development at JoVE.com

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

上皮細胞の再増殖および腎組織の開発のための齧歯類細胞外マトリックスの足場の調製

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

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Research

JoVE Journal

Bioengineering

15.9K ビュー.  Northwestern University. This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.