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.

<ol>
	<li>Uzarski, J. S., Xia, Y., Belmonte, J. C., Wertheim, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+strategies+in+kidney+regeneration+and+tissue+engineering.">New strategies in kidney regeneration and tissue engineering.</a> Current opinion in nephrology and hypertension. 23, 399-405 (2014).</li><li>Soto-Gutierrez, A., Wertheim, J. A., Ott, H. C., Gilbert, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+whole-organ+assembly:+moving+toward+transplantation+on+demand.">Perspectives on whole-organ assembly: moving toward transplantation on demand.</a> J Clin Invest. 122, 3817-3823 (2012).</li><li>Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+allogeneic+and+xenogeneic+tissue+as+a+bioscaffold+for+regenerative+medicine:+factors+that+influence+the+host+response.">Decellularized allogeneic and xenogeneic tissue as a bioscaffold for regenerative medicine: factors that influence the host response.</a> Ann Biomed Eng. 42, 1517-1527 (2014).</li><li>Miner, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+basement+membrane+components.">Renal basement membrane components.</a> Kidney Int. 56, 2016-2024 (1999).</li><li>Nichol, J. W., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+Tissue+Engineering:+Engineering+Biological+Tissues+from+the+Bottom+Up.">Modular Tissue Engineering: Engineering Biological Tissues from the Bottom Up.</a> Soft matter. 5, 1312-1319 (2009).</li><li>Ott, H. 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=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat Med. 14, 213-221 (2008).</li><li>Caralt, M., 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=Optimization+and+critical+evaluation+of+decellularization+strategies+to+develop+renal+extracellular+matrix+scaffolds+as+biological+templates+for+organ+engineering+and+transplantation.">Optimization and critical evaluation of decellularization strategies to develop renal extracellular matrix scaffolds as biological templates for organ engineering and transplantation.</a> American Journal of Transplantation. 15, 64-75 (2015).</li><li>Badylak, S. F., Taylor, D., Uygun, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-organ+tissue+engineering:+decellularization+and+recellularization+of+three-dimensional+matrix+scaffolds.">Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix scaffolds.</a> Annu Rev Biomed Eng. 13, 27-53 (2011).</li><li>Orlando, G., 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=Discarded+human+kidneys+as+a+source+of+ECM+scaffold+for+kidney+regeneration+technologies.">Discarded human kidneys as a source of ECM scaffold for kidney regeneration technologies.</a> Biomaterials. 34, 5915-5925 (2013).</li><li>Sullivan, D. 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. J., 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=Regeneration+and+experimental+orthotopic+transplantation+of+a+bioengineered+kidney.">Regeneration and experimental orthotopic transplantation of a bioengineered kidney.</a> Nat Med. 19, 646-651 (2013).</li><li>Bonandrini, B., 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=Recellularization+of+well-preserved+acellular+kidney+scaffold+using+embryonic+stem+cells.">Recellularization of well-preserved acellular kidney scaffold using embryonic stem cells.</a> Tissue Eng Part A. 20, 1486-1498 (2014).</li><li>Burgkart, R., 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=Decellularized+kidney+matrix+for+perfused+bone+engineering.">Decellularized kidney matrix for perfused bone engineering.</a> Tissue Eng Part C Methods.2. 20, 553-561 (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=Mouse+stem+cells+seeded+into+decellularized+rat+kidney+scaffolds+endothelialize+and+remodel+basement+membranes.">Mouse stem cells seeded into decellularized rat kidney scaffolds endothelialize and remodel basement membranes.</a> Organogenesis. 8, 49-55 (2012).</li><li>Uzarski, J. S., 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=Dual-purpose+bioreactors+to+monitor+non-invasive+physical+and+biochemical+markers+of+kidney+and+liver+scaffold+recellularization.">Dual-purpose bioreactors to monitor non-invasive physical and biochemical markers of kidney and liver scaffold recellularization.</a> Tissue Eng Part C Methods. , (2015).</li><li>Brien, J., Wilson, I., Orton, T., Pognan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+Alamar+Blue+(resazurin)+fluorescent+dye+for+the+assessment+of+mammalian+cell+cytotoxicity.">Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity.</a> European journal of biochemistry / FEBS. 267, 5421-5426 (2000).</li><li>Loghman-Adham, M., Nauli, S. M., Soto, C. E., Kariuki, B., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+epithelial+cells+from+human+autosomal+dominant+polycystic+kidney+cysts.+American+journal+of+physiology.">Immortalized epithelial cells from human autosomal dominant polycystic kidney cysts. American journal of physiology.</a> Renal physiology. 285, F397-F412 (2003).</li></ol>

The authors have nothing to disclose.

Il protocollo descritto decellularization produce costantemente una ECM rene completamente acellulare che serve da modello 3D per coltura di cellule umane renali corticali tubulari epiteliali (sia prossimale e distale del tubulo-derivato), in aggiunta a cellule endoteliali vascolari 7,17. Il sistema vascolare renale cannulata serve come elemento chiave per la consegna uniforme dei reagenti e cellule in tutto il ponteggio all&#39;interno di un bioreattore set-up, permettendo così la perfusione decellularizati...

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

Poiché il numero di pazienti affetti da insufficienza renale all&#39;ultimo stadio continua ad aumentare, vi è una carenza grave e crescente nel numero di donatori reni disponibili per il trapianto. L&#39;incapacità di soddisfare la richiesta di un numero sempre crescente di candidati wait-vendita per il trapianto di rene ha spinto la ricerca in ingegneria organo rene con l&#39;obiettivo finale di sviluppare su misura, innesti renali impiantabili su richiesta 1,2. Costruire funzionante tessuto renale dalle stesse cellule del paziente eliminerebbe la necessità di immunosoppressione per tutta la vita, diminuire la quantità di tempo che i pazienti trascorrono in dialisi in attesa di un trapianto di rene, ed estendere il trapianto salvavita a più pazienti con malattia renale cronica. Il primo passo verso bioingegneria un tessuto renale utilizzando cellule derivate da pazienti è quello di sviluppare un patibolo che funge da substrato di supporto per parenchima renale (ad es epithe tubolareLIAL), stroma fibroblasti, e la crescita delle cellule vascolari. Ponteggi biomateriale derivati ​​da organi naturali matrici extracellulari (ECM) hanno diverse caratteristiche che li rendono auspicabile per l&#39;uso in ingegneria dei tessuti, compresa la loro composizione naturale biologico; macro e microstruttura opportuno dotare funzione fisiologica; e biocompatibilità cellulare, promuovendo l&#39;adesione cellulare, la migrazione e il tessuto costruttivo rimodellamento 3. Un metodo promettente per la produzione di scaffold per la rigenerazione dei tessuti renali è attraverso decellularization di allogenici o xenogenici reni che conserva gran parte della complessa composizione proteina naturale della ECM rene 4, conservano la complessità architettonica intrinseca dell&#39;organo, e superare le difficoltà associate con bottom up ingegneria dei tessuti cellularized spessore, fornendo un apporto vascolare alle cellule in via di sviluppo dopo patibolo Recellularization 5. Perfusione decellularization è un processo inquali detergenti, enzimi, o altre soluzioni cellule interrompendo sono uniformemente forniti tramite la rete vascolare dell&#39;organo 6. Questa strategia è stata stabilita come un processo efficiente per ricavare acellulare ponteggi ECM organo-based come tridimensionale (3D), modelli biologici per l&#39;ingegneria intero organo 6-8, come dimostra lo sviluppo di modelli renali acellulari da scartati reni umani 9 e reni xenogeniche ottenuti da grande animale (ad esempio maiale 10, capra 11) e le fonti roditori 12. In particolare, l&#39;uso di piccoli modelli animali come roditori richiede meno cellule e terreni di coltura, che è particolarmente utile per gli studi di organo Recellularization in cui i numeri di cellule sono in genere limitate, come è il caso con i tessuti derivati ​​dalle cellule staminali. L&#39;obiettivo del protocollo decellularization descritto è quello di produrre un ECM renale acellulare che può essere utilizzato come sistema di ponteggio 3D per la rigenerazione di structur renees, tra cui tubuli nefrone che si ripopolò nel presente esempio con renale corticale epiteliali tubulare umano (RCTE), le cellule. Abbiamo precedentemente descritto la nostra rigorosa valutazione di un ottimale, basato detergente protocollo ratto decellularization rene 7, che è più rapida (circa un giorno) rispetto ad altri metodi in precedenza segnalati (Ross et al .- 5 giorni 12: Song et al .- 4,5 giorni 13), ed espone l&#39;organo ad una concentrazione notevolmente inferiore (0,1%) del solfato di sodio dodecil denaturante (SDS) durante decellularization di relazioni precedenti 12-15. Un numero limitato di studi hanno descritto l&#39;uso di reni roditori per decellularization e il successivo utilizzo come impalcatura 3D per il ripopolamento cellulare (recensione altrove 1) 12-16. In questo protocollo, forniamo una descrizione dettagliata della nostra precedentemente stabilito, la strategia ottimale per la produzione di ponteggi decellularization renali acellularida Sprague Dawley reni di ratto 7. Utilizzando bioreattori a perfusione progettati su misura in grado di doppia semina e manutenzione perfusione cultura 17, abbiamo recellularize i ponteggi renali acellulari con le cellule umane RCTE, che, sistematicamente, ripopolare il tubolare in queste matrici decellularized, proliferano, e sopravvivono nella cultura perfusione per oltre una settimana. Si dimostra ulteriormente il nostro uso del test resazurina perfusione - un poco costoso, non citotossica e valutazione metabolica non invasivo precedentemente utilizzato per studi citotossicità 17 - fornire un&#39;indicazione della vitalità cellulare e la proliferazione nei reni recellularized nel tempo 7.

<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

Dettagli Questo protocollo la generazione di acellulare, ancora biofunzionali, renali matrice extracellulare (ECM) ponteggi che sono utili come piccoli substrati del modello di sviluppo tessuto dell&#39;organo scala. Sprague Dawley reni di ratto sono cannulati inserendo un catetere nell&#39;arteria renale e perfusi con una serie di detergenti bassa concentrazione (Triton X-100 e sodio dodecil solfato (SDS)) su 26 hr derivare intatte, impalcature intero renali con intatta vascolarizzazione perfusable, glomeruli e tubuli renali. Dopo decellularization, l&#39;impalcatura renale si deposita in un recipiente perfusione bioreattore progettati su misura, e l&#39;arteria renale cateterizzato è collegato ad un circuito di perfusione costituito da: una pompa peristaltica; tubi; e sonde opzionali per pH, ossigeno disciolto, e la pressione. Dopo la sterilizzazione il ponteggio con acido peracetico ed etanolo, e del bilanciamento del pH (7,4), il ponteggio rene viene preparato per la semina tramite perfusione di terreno di coltura in un large capacità incubatore mantenuto a 37 ° C e 5% CO 2. Quaranta milioni corticale epiteliali tubolari (RCTE) cellule renali vengono iniettate attraverso l&#39;arteria renale e rapidamente perfusi attraverso scaffold sotto alto flusso (25 ml / min) e pressione (~ 230 mmHg) per 15 min prima di ridurre la portata ad una frequenza fisiologica (4 ml / min). Cellule RCTE popolano soprattutto la ECM nicchia tubolare all&#39;interno della corteccia renale, proliferano e formano strutture epiteliali tubulari oltre sette giorni di coltura perfusione. Una soluzione resazurina 44 micron in terreno di coltura è perfuso attraverso il rene per 1 ora durante gli scambi medi per fornire un fluorimetrico, basato redox-valutazione metabolica di vitalità e proliferazione cellulare durante tubulogenesi. Il bioreattore perfusione renale consente campionamento non invasivo di media per la valutazione biochimica, e molteplici luci di immissione permettono semina retrograda alternativo attraverso la vena renale o dell&#39;uretere. Questi protocolli possono essere usati per recellularize scaffold renali convarietà di tipi cellulari, tra cui cellule endoteliali vascolari, epiteliale tubulare, e fibroblasti stromali, per la valutazione rapida all&#39;interno di questo sistema.

I reni sequenzialmente perfusi con acqua e diluire soluzioni detergenti (1% Triton X-100, 0,1% SDS) secondo una precedentemente stabilito, protocollo ottimale decellularization (vedere Figura 1A, B) 7, diventa progressivamente più trasparente su un periodo di 26 ore, come mostrato nella Figura 2A. La risultante impalcatura rene acellulare è privo di cellule e mantiene un ECM renale coesa supportato da una capsula renale intatta, che è intatto seguendo il protocollo di perf...

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.

Ripopolamento cellule epiteliali e preparazione di roditori matrice extracellulare Ponteggi per lo sviluppo del tessuto renale

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

epithelial-cell-repopulation-preparation-rodent-extracellular-matrix

Research

JoVE Journal

Bioengineering

15.9K Views.  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.