Name: procedure for decellularization of porcine heart by retrograde coronary perfusion
Uploaded: 2012-12-06T12:00:00
Description: Watch this Scientific Journal Video about Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion at JoVE.com

Forfatterne vil gerne anerkende Brogan Guest, Michelle Weaver, og Kristen Lippert. Finansieringen af ​​denne undersøgelse blev leveret af NIH Grant R03EB009237 samt NIH uddannelsesstipendierne T32EB001026-06 fra National Institute of Biomedical Imaging And Bioengineering og T32HL076124-05.

1. Tissue Forberedelse og Opsætning af forsøg <ol><li> Harvest svin orgel umiddelbart efter eutanasi fra et slagteri eller forskningsanlæg og skylles overskydende blod. Trim hjertet af overskydende fedt og væv, holde forkamre og aorta intakt. Fraklippe fedtstof at adskille lungepulsåren fra aorta. Hvis der er nogen nedskæringer i vævet, kasseres korrekt. </li><li> Wrap hver hjertet individuelt i fryseren papir og gemme alle væv i en -80 ° C fryser i mindst 24 timer for at sikre fuldstændig frysning. </li><li...

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Den aktuelle undersøgelse beskrevne metode for en konsekvent og effektiv decellularization af et svin hjerte. Protokollen var en modifikation til en tidligere udgivet rapport 1, og omfattede længere udsættelse for flow og øget pres, som gav mere reproducerbare resultater. Den resulterende decellulariseret væv mødte alle de offentliggjorte kriterier for en vellykket decellularization af væv 2. Hyppige opløsning ændringer blev udført for at begrænse genindførelse af cellulært materiale t...

<table border="1"><tbody><tr><td> Navn på reagens / Material </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Trypsin </td><td> Gibco </td><td> 15.090 </td><td></td></tr><tr><td> EDTA </td><td> Fisher </td><td> BP120-500 </td><td></td></tr><tr><td> NaN3 </td><td> Sigma </td><td> S2002-500G </td><td></td></tr><tr><td> Triton X-100 </td><td> Sigma </td><td> X100-1L </td><td></td></tr><tr><td> 10X PBS </td><td> Fisher </td><td> BP399-20 </td><td></td></tr><tr><td> Natriumdeoxycholat </td><td> Sigma </td><td> D6750-500G </td><td></td></tr><tr><td> Pereddikesyre </td><td> Pfaltz og Bauer </td><td> P05020 </td><td> 35% CAS # 79-21-0 </td></tr><tr><td> Ethanol </td><td> Pharmco </td><td> 111000200 </td><td></td&gt; </tr><tr><td> Masterflex Pump Drive </td><td> Cole Parmer </td><td> SI-07524-50 </td><td></td></tr><tr><td> Masterflex Tubing </td><td> Cole Parmer </td><td> 96.400-18 </td><td> Størrelse 18 </td></tr><tr><td> Barbed Reducer </td><td> Cole Parmer </td><td> EW-30612-20 </td><td></td></tr><tr><td> 4L Beaker </td><td> Fisher Scientific </td><td> 02-540T </td><td></td></tr></tbody></table>

procedure for decellularization of porcine heart by retrograde coronary perfusion

Perfusion baseret helt organ decellularization har for nylig fået interesse inden for tissue engineering som et middel til at skabe stedspecifikke ekstracellulære matrix scaffolds, mens de stort set bevarer den native struktur af stilladset. Til dato er denne fremgangsmåde blevet anvendt i en række forskellige organsystemer, herunder hjerte, lunge og lever 1-5. Tidligere decellularization metoder for væv uden en let tilgængelig vaskulære netværk har påberåbt langvarig udsættelse af væv til opløsninger af vaske-og rengøringsmidler, syrer eller enzymatiske behandlinger som et middel til at fjerne de cellulære og nukleare komponenter fra det omgivende ekstracellulære miljø 6-8. Imidlertid effektiviteten af ​​disse metoder hængslet af evnen af ​​opløsningerne gennemtrænger vævet ved diffusion. I modsætning hertil effektivt perfusion af organer gennem naturlige vaskulære system reducerede diffusionsafstanden og lettet transport af decellularizatipå midler ind i vævet og cellulære komponenter ud af vævet. Heri, beskriver vi en fremgangsmåde til fuldt decellularize et intakt porcin hjerte gennem koronar retrograd perfusion. Protokollen gav et fuldt decellulariseret hjerte ekstracellulære matrix (c-ECM) stillads med den tredimensionale struktur af hjertet intakt. Vores metode en række enzymer, detergenter og syrer kombineret med hypertoniske og hypotonisk skylninger for at hjælpe med lysering og fjernelse af celler. Protokollen anvendt en trypsinopløsning at frigøre celler fra matrixen efterfulgt af Triton X-100 og natriumdeoxycholat opløsninger til hjælp ved fjernelse af cellemateriale. Den beskrevne protokol anvender også perfusion hastigheder på over 2 L / min i længere tid. Den høje strømningshastighed, kombineret med opløsning ændres tilladt transport af midler til vævet uden forurening af cellerester og sikret en effektiv skylning af vævet. Den beskrevne metode fjernede alt nukleart materiale fra Native porcint hjertevæv, hvilket skaber en stedspecifik hjerte ECM stillads, der kan anvendes til en lang række applikationer.

Virkningen af ​​decellularization på hele porcine hearts naturligvis varierer på grund af forskelle i størrelse, tryk og beholderen arrangement. Derfor vil den nøjagtige sammensætning af de afledte ekstracellulære matrix stilladser ikke være det samme fra hjerte til hjerte. Færdiggørelsen af ​​den beskrevne protokol, vil give et hjerte, der vises hvid eller gennemsigtig, hvilket indikerer tab af cellulært materiale. Det er imidlertid almindeligt accepteret, at et væv kan betragtes som &quot;decellular...

Watch this Scientific Journal Video about Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion at JoVE.com

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

En fremgangsmåde til hurtigt og fuldstændigt fjerne cellulære bestanddele fra en intakt porcin hjerte via retrograd perfusion beskrives. Denne metode giver en stedsspecifik hjerte ekstracellulær matrix stillads, der har potentiale til brug i flere kliniske anvendelser.

Procedure for Decellularization af porcin Heart ved retrograd Coronarperfusionstrykket

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

Research

JoVE Journal

Bioengineering

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