Name: procedure for decellularization of rat livers in an oscillating-pressure perfusion device
Uploaded: 2015-08-10T13:00:00
Description: Watch this Scientific Journal Video about Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device at JoVE.com

The authors would like to gratefully thank Steffen Lippert, Khalid Aliyev, Korinna J&#246;hrens and Katharina Struecker for their help during this project.

Djuren hålls vid anläggningen för Experimental Medicine (FEM, Charité, Berlin, Tyskland), och alla experimentella protokoll har granskats och godkänts av staten Office of Health och lokala frågor (LAGeSo, Berlin, Tyskland,. Dnr O 0365 / 11). 1. Lever Skörd <ol><li> Pre-kirurgisk Framställning <ol><li> Använd en korkplatta för fixering. Sätt en Medicinsk duk på plattan. Med hjälp av fyra nålar, fixa en inandningsmask på korken platta för intraoperativ inandning narkos. </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=An+overview+of+tissue+and+whole+organ+decellularization+processes.">An overview of tissue and whole organ decellularization processes.</a> Biomaterials. 32, 3233-3243 (2011).</li><li>Wang, X., 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+liver+scaffolds+effectively+support+the+proliferation+and+differentiation+of+mouse+fetal+hepatic+progenitors.">Decellularized liver scaffolds effectively support the proliferation and differentiation of mouse fetal hepatic progenitors.</a> J Biomed Mater Res A. , (2013).</li><li>Uygun, B. E., 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=Organ+reengineering+through+development+of+a+transplantable+recellularized+liver+graft+using+decellularized+liver+matrix.">Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.</a> Nat Med. 16, 814-820 (2010).</li><li>Shupe, T., Williams, M., Brown, A., Willenberg, B., Petersen, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+decellularization+of+intact+rat+liver.">Method for the decellularization of intact rat liver.</a> Organogenesis. 6, 134-136 (2010).</li><li>Bao, 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=Construction+of+a+portal+implantable+functional+tissue-engineered+liver+using+perfusion-decellularized+matrix+and+hepatocytes+in+rats.">Construction of a portal implantable functional tissue-engineered liver using perfusion-decellularized matrix and hepatocytes in rats.</a> Cell transplantation. 20, 753-766 (2011).</li><li>Baptista, P. 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=The+use+of+whole+organ+decellularization+for+the+generation+of+a+vascularized+liver+organoid.">The use of whole organ decellularization for the generation of a vascularized liver organoid.</a> Hepatology. 53, 604-617 (2011).</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>Soto-Gutierrez, 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=A+whole-organ+regenerative+medicine+approach+for+liver+replacement.">A whole-organ regenerative medicine approach for liver replacement.</a> Tissue engineering. Part C, Methods. 17, 677-686 (2011).</li><li>De Kock, 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=Simple+and+quick+method+for+whole-liver+decellularization:+a+novel+in+vitro+three-dimensional+bioengineering+tool.">Simple and quick method for whole-liver decellularization: a novel in vitro three-dimensional bioengineering tool.</a> Archives of toxicology. 85, 607-612 (2011).</li><li>Park, K. M., Woo, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+decellularization+for+multi-organ+scaffolds+in+rats.">Systemic decellularization for multi-organ scaffolds in rats.</a> Transplantation proceedings. 44, 1151-1154 (2012).</li><li>Shirakigawa, N., Ijima, H., Takei, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+liver+as+a+practical+scaffold+with+a+vascular+network+template+for+liver+tissue+engineering.">Decellularized liver as a practical scaffold with a vascular network template for liver tissue engineering.</a> J Biosci Bioeng. , (2012).</li><li>Ren, H., 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=Evaluation+of+two+decellularization+methods+in+the+development+of+a+whole-organ+decellularized+rat+liver+scaffold.">Evaluation of two decellularization methods in the development of a whole-organ decellularized rat liver scaffold.</a> Liver Int. 33, 448-458 (2013).</li><li>Struecker, 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=Improved+rat+liver+decellularization+by+arterial+perfusion+under+oscillating+pressure+conditions.">Improved rat liver decellularization by arterial perfusion under oscillating pressure conditions.</a> J Tissue Eng Regen Med. , (2014).</li><li>Struecker, 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=Porcine+Liver+Decellularization+Under+Oscillating+Pressure+Conditions:+A+Technical+Refinement+to+Improve+the+Homogeneity+of+the+Decellularization+Process.">Porcine Liver Decellularization Under Oscillating Pressure Conditions: A Technical Refinement to Improve the Homogeneity of the Decellularization Process.</a> Tissue engineering. Part C, Methods. , (2014).</li></ol>

Även den presenterade tekniken för råttlever skörd och decellularisering är lätt reproducerbar, det finns vissa viktiga steg att tänka på: Under förberedelse för lever skörd, är det viktigt att undvika allvarlig blödning, eftersom det kommer att aktivera blodkoagulation och kan leda till blodproppsbildning i levern. Enligt vår mening, är det fördelaktigt att incisionsfilm bukaorta direkt före kanyle av portalen ven för att undvika blodinflöde via leverartären under perfus...

Decellularisering och recellularization kan möjliggöra bildandet av funktionella, transplanterbara organ in vitro 1. Genom att avlägsna celler och antigent material (t ex., DNA, a-Gal-epitoper) från ett organ, den icke- eller mindre immunogena extracellulär matrix (ECM) kan erhållas. Denna matris bevarar den tredimensionella mikroanatomi av ett organ och kan tjäna som en idealisk biomatris för rekolonisering med celler från en annan, möjligen xenogena ursprung 2. Således kan en decellulariserad råttlever matrisen nyinsatta med humana leverceller. Denna humaniserade mikro levern skulle kunna tjäna som en ex vivo-modell för forskning om sjukdomar (t.ex.., Medfödda ämnesomsättningssjukdomar, virussjukdomar eller maligniteter) eller preklinisk läkemedelstestning 3. Flera olika protokoll för råttlever perfusion decellularisering har redan publicerats 4-13. I alla protokoll, decellularsering uppnåddes genom perfusion av alkaliska joniska eller icke-joniska detergenter via kanyle portvenen. Så vitt vi vet var vi den första gruppen att rapportera råttlever decellularisering genom selektiv perfusion via portalen ven och / eller råtta leverartären 14. Aktivera selektiv perfusion av de olika vaskulära system i levern kan möjliggöra bättre decellularisering resultaten och, dessutom, kan spela en viktig roll i cellulär rekolonisering. I studien beskrivs här, var lever perfusion i en skräddarsydd egen perfusion enhet möjliggör perfusion enligt oscillerande tryckförhållanden. Dessa tryckförhållanden härma den fysiologiska andningsberoende perfusion av levern: in situ, hänger levern under copula av membranet, vars rörelse under andning har en direkt inverkan på leverperfusion. Inspiration leder specifikt till sänkning av membranet och klämma av levern, optimeraLever och venösa utflödet leder medan utgången av förhöjda lever och sänkning av intraabdominella trycket att optimera portal venös inflöde 15. Vårt mål var att utvärdera om oscillerande tryckförhållanden har en inverkan på homogeniteten av råttlever perfusion decellularisering genom att härma intraabdominal villkor ex vivo. Den homogenitet decellularisering processen kan vara en underskattad faktor i perfusion decellularisering. Alla kända medel som används för lever decellularisering orsaka förändringar av ECM. Celler i dåligt perfunderade områden kvar inom ECM, medan andra områden är redan helt decellulariseras. För att lösa de kvarvarande cellerna, måste perfusion varaktighet eller tryck höjas, vilket flera förändringar till väl perfusion områden. Därför bör tvättmedel för decellularisering fördelas homogent inom organet.

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			<td></td>
			<td></td>
			<td></td>
			<td>Self built arterial cannula</td>
		</tr>
		<tr>
			<td>&#160;Portex Non Sterile Polyethene Tubing</td>
			<td>SIMS Portex</td>
			<td>REF 800/110/100</td>
			<td>0,28mm ID 0,61mm OD</td>
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		<tr>
			<td>&#160;Portex Non Sterile Polyethene Tubing</td>
			<td>SIMS Portex</td>
			<td>REF 800/110/200</td>
			<td>0,58mm ID 0,96m OD</td>
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		<tr>
			<td>Venodrop Safe butterfly catheter</td>
			<td>Fresenius Kabi</td>
			<td>3275851</td>
			<td>21 G</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>portal vein cannula</td>
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		<tr>
			<td>Periphereal Venous Catheter</td>
			<td>BD</td>
			<td>393224</td>
			<td>BD Venflon Pro 20G</td>
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		<tr>
			<td>three-way stopcock</td>
			<td>smiths medical</td>
			<td>888-101RE</td>
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			<td></td>
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			<td>surgery</td>
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			<td>Cotton Sticks</td>
			<td>Hecht-Assistent</td>
			<td>4302</td>
			<td></td>
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			<td>Cotton Pads&#160;</td>
			<td>Shaoxing Zhengde Surgical dressing</td>
			<td>13H118-03</td>
			<td></td>
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		<tr>
			<td>Gauze Bandage</td>
			<td>Hubei Haige&#160; Medical Instruments</td>
			<td>14388</td>
			<td></td>
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		<tr>
			<td>&#160;Ringer Solution</td>
			<td>Fresenius Kabi</td>
			<td>13 HKP022</td>
			<td>1000ml</td>
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		<tr>
			<td>10ml Syringe</td>
			<td>Braun</td>
			<td>4606108V</td>
			<td>10ml/ Luer Solo</td>
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		<tr>
			<td>5ml Syringe</td>
			<td>Braun</td>
			<td>4606061V</td>
			<td>5ml /Luer Solo</td>
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		<tr>
			<td>Suture (Silk 6/0)</td>
			<td>Resorba</td>
			<td>H1F</td>
			<td>LOT 105001.81</td>
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			<td>medical drape</td>
			<td>Shaoxing Zhengde Surgical dressing</td>
			<td>D0613011</td>
			<td></td>
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			<td></td>
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			<td>surgical instruments</td>
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			<td>needle holder</td>
			<td>Geuder</td>
			<td>17570</td>
			<td></td>
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			<td>micro-forceps</td>
			<td>Inox-Electronic</td>
			<td>91150-20</td>
			<td></td>
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			<td>micro-scissors</td>
			<td>Martin</td>
			<td>11-740-11</td>
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			<td>micro-forceps</td>
			<td>S&#38;T&#160;</td>
			<td>112314</td>
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			<td>Clamp</td>
			<td>Aesculap</td>
			<td>BH111R</td>
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			<td>scissors</td>
			<td>F S T&#160;</td>
			<td>14501-14</td>
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			<td>surgical forceps</td>
			<td>Aesculap</td>
			<td>BD 557</td>
			<td></td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Decellularisation</td>
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		<tr>
			<td>Respirator</td>
			<td>Resmed</td>
			<td>14.24.11.0004</td>
			<td>SmartAIR ST</td>
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		<tr>
			<td>Perfusion Device</td>
			<td>Charite, medical engineering laboratory</td>
			<td>custome-made device</td>
			<td>decellularisation device</td>
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			<td>peristaltic pump ismatec reglo ICC</td>
			<td>IDEX</td>
			<td>ISM4408&#160;</td>
			<td>4-channel</td>
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			<td>heidelberger extension 75 cm&#160;</td>
			<td>Fresenius Kabi</td>
			<td>2873</td>
			<td>75 cm</td>
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			<td>MS/CA pump-segment</td>
			<td>IDEX</td>
			<td>IS 3510&#160;</td>
			<td>MS/CA/click&#39;n&#39;go/POM-C</td>
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		<tr>
			<td>CA 2-stopper tube</td>
			<td>Pharmed</td>
			<td>BPT NSF-51</td>
			<td></td>
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		<tr>
			<td>bubble trap&#160;</td>
			<td></td>
			<td>custome-made item</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock hose connector</td>
			<td>Neolab</td>
			<td>No. 02-1887</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Detergents</td>
		</tr>
		<tr>
			<td>SDS pellets</td>
			<td>Carl Roth&#160;</td>
			<td>CN30.4&#160;</td>
			<td>2,5 kg</td>
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		<tr>
			<td>Triton X-100</td>
			<td>Carl Roth&#160;</td>
			<td>3051.1&#160;</td>
			<td>10l</td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>DPBS</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>staining</td>
		</tr>
		<tr>
			<td>Eosin 1%</td>
			<td>Morphisto</td>
			<td>10177</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer hematoxylin</td>
			<td>AppliChem</td>
			<td>A4840</td>
			<td></td>
		</tr>
		<tr>
			<td>gomori staining</td>
			<td>Morphisto</td>
			<td>11104</td>
			<td></td>
		</tr>
		<tr>
			<td>AlcainBlue-PAS staining</td>
			<td>Morphisto</td>
			<td>11388</td>
			<td></td>
		</tr>
		<tr>
			<td>Direct Red 80&#160;</td>
			<td>Sigma Aldrich</td>
			<td>365548</td>
			<td></td>
		</tr>
	</tbody>
</table>

procedure for decellularization of rat livers in an oscillating-pressure perfusion device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Den homogenitet och därmed effektiviteten av olika decellularisering protokoll utvärderades genom makroskopisk observation, histologisk analys och analys av den återstående DNA-innehållet i decellulariserade lever matriser. Vidare korrosionsgjutning utförs för att visualisera den intakta mikroanatomi i lever efter decellularisering. Makroskopi Under decellularisering, levrar blir lucent, vilket indikerar avlägsnande av cellulärt innehåll. Lever perfusio...

Watch this Scientific Journal Video about Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device at JoVE.com

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Förfarande för decellularisering av råttlevrar i ett oscillerande tryck perfusionsanordning

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

Research

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Bioengineering

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The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

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