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

Hayvanlar Deneysel Tıp (FEM, Charité, Berlin, Almanya) için Tesisi&#39;nde tutuldu ve tüm deneysel protokoller gözden geçirilecek ve Devlet Sağlık ve Yerel İşleri Ofisi (LAGeSo, Berlin, Almanya tarafından kabul edildi;. Reg No O 0365 / 11). 1. Karaciğer Hasat <ol><li> Ön Cerrahi hazırlık <ol><li> Sabitleme için bir mantar plaka kullanın. Plaka üzerinde tıbbi örtü koyun. Dört iğneler kullanılarak, intraoperatif inhalasyon narkoz için mantar plaka üzerinde bir inhalasyon maskesi düzeltmek. </li></ol></li><li> Portal Venöz Kanül prearrangement <ol><li> Üç-yollu bir periferal venöz kateter (G 20) bağlanır. kateter yaklaşık bir uzunluğa sahip, eğik olarak kesilmelidir. 1,5 cm. </li><li> De hava Ringer çözeltisi ile doldurulmuş bir 10 ml şırınga kullanarak sistem. Dikkatle de hava gaz embolisi olumsuz perfüzyon decellularization etkileyebilir çünkü çok önemlidir. </li></ol></li><li> PrearrangementArter Kanül <ol><li> Kelebek kanül kullanıldığı ve 5 mm bir uzunluğa iğne kesti. 5 mm iğne ve superglue ile sabitleyin, 5 cm tüp (OD 0.96 mm ID 0.58 mm) takınız. Büyük tüp içine ve superglue ile sabitleyin, 2 cm uzunluğunda bir tüp (OD 0.61 mm ID 0.28 mm) yerleştirin. </li><li> Bir dayanak olarak bu yapı üzerinde başka tüp kayma ve superglue ile sabitleyin. De hava Ringer çözeltisi ile dolu bir 5 ml şırıngaya arteryel kanül. </li></ol></li><li> Anestezi ve Analjezi <ol><li> 1.5 L / dakikalık bir akış oranında% 3.5 oksijen izofluran anestezi indüksiyon odasında sıçan narkozu neden. </li><li> Çalışma sırasında analjezi sağlamak için, karın içinden metamizol (100 mg / kg vücut kütlesi) ve düşük doz ketamin / medetomidini (kg vücut ağırlığı başına 10 mg / 0.1 mg) enjekte edilir. </li><li> Cerrahi anestezi sonraki bakım için inhalasyon maskesi üzerinden 1 L / dk oksijen% 1.5 izofluran sağlayın. </li></ol></li><li> Ameliyat öncesi Düzenlemeler &lt;ol&gt; <li> Elektrikli kesme makinesi ile liberal karın Tıraş. Dezenfeksiyonu için% 70 etanol ile karın traş kesi sitesini nemlendirin. </li><li> Hazırlanan plaka üzerinde yatar pozisyonda hayvan yerleştirin inhalasyon maskesi içine kafasını takın ve tıbbi bant ve iğneler ile bacaklarda düzeltmek. Bir gazlı bez kompres ile aşırı etanol ve kürk çıkarın. </li></ol></li><li> Anestezi Değerlendirmesi <ol><li> Solunum hızı izleyerek anestezi derinliği değerlendirin. Anestezi (40-60 solunum / dk) yeterince derin görünüyor zaman analjezi her iki arka bacaklarda ayak parmakları arasında cilt çimdik cerrahi forseps kullanarak ayak tutam refleks tetiklemek için deneyerek yeterli olup olmadığını kontrol edin. </li></ol></li><li> Karın Yaklaşım <ol><li> Mesane üstünde kaudal karın duvarı içine medyan kesi yapmak. V-şekilli bir biçimde her iki lateral kot kemerler için, bu noktadan itibaren kesilir. Diyaframın içine kesmek için özen gösterin. Göğüs üzerine kesilmiş karın duvarı çekin. </li&gt; <li> Bir Overholt kelepçe ile kılıç şeklinde Fix cranially çekin ve bir lastik bant ile sabitleyin. Bağırsak tahliye ıslak pamuk bezlerden kullanın. Islak gazlı bez içinde bağırsak sarın. Ayrıca, ıslak bandaj ile tüm karın marjlarını kapsamaktadır. </li></ol></li><li> Karaciğer Hazırlık <ol><li> Falciform ligament parçalara ayır. Cranially diyaframın kubbe altında medial ve sol karaciğer loblar tutmak için ıslak bir gazlı bez bandaj kullanın. Triadın ve üst omentum karaciğer lobu Açığa. </li><li> Lob mobil kadar dikkatlice üst omentum lob bağlı ligament incelemek için iki microforceps ve yaylı makas kullanın. Yerinde medial ve sol lobu tutan, gazlı bez kompres altında ıslak pamuklu çubuklarla kullanarak lob hareket ettirin. </li><li> Üst omentum karaciğer lobunun seferber sonra sola mide hareket ettirin. Bir kelepçe ile mide Fix ve microforceps ve yaylı makas ile küçük omentum teşrih. </li><li> Alt omenta Seferberl karaciğer lobu mobil ve daha sonra onun boşluğuna geri lobun taşımak kadar. Duodenum korumak için bir Backhaus kelepçe kullanın. Streç ve triadın maruz sola duodenum taşıyın. Gergin hepatoduodenal ligaman koledok circumscribe. </li><li> Adipoz ve pankreas dokusundan safra kanalını parçalara ayır. Safra kanalını safra kanalı bifurkasyondan 1,5 cm kesin. Çevreleyen yağ dokusundan portal ven parçalara ayır. Gastroduodenal ven Açığa. </li><li> Ipek 6-0 ile iki kez gastroduodenal ven bağlanır ve 2 bitişik harfler arasındaki damar bölün. Tüm arteriyel rami ortaya kadar çevreleyen dokudan çölyak gövde ve ortak hepatik arter parçalara ayır. </li><li> Çevreleyen dokudan gastroduodenal arter Özgürleştirin. Birbirinden uzak bağları, ipek 6-0 ile iki kez gastroduodenal arter kravat. 2 bitişik harfler arasındaki gastroduodenal arter parçalara ayır. </li><li> Dalak arteri kurtarmakçevreleyen dokudan. Birbirinden uzak bağları, ipek 6-0 ile iki kez dalak arteri bağla. 2 bitişik harfler arasındaki dalak arteri parçalara ayır. </li><li> Çevreleyen doku sol gastrik arter Özgürleştirin. Birbirinden uzak bağları, ipek 6-0 ile iki kez sol gastrik arter kravat. 2 bitişik harfler arasındaki arter parçalara ayır. </li><li> Sağ böbrekte ve sağ yanal karaciğer lobu arasındaki alt kaval ven circumscribe. Retroperitoneal uzaydan damar parçalara ayır. Çölyak gövde izleyerek aorta Açığa. </li><li> Retroperitoneal adipoz doku teşrih. Inferior vena kava içine 1 ml serum fizyolojik içinde 1.000 IE heparin enjekte edilir. Kanül geri çekin ve pamuklu ped ile kesi kapatın. Heparin sistemik etki için 1-2 dakika bekleyin. </li><li> Forseps ile aorta circumscribe. Aorta incelemek ve sıçan asıl bulbus olfaktoryusları alındı. Ayrıca, böbrek distal inferior vena kava teşrih. </li></ol></li><li> Portal Venöz Kanülasyon (Kullanım bölüm 1.2 hazırlanan kanül) <ol><li> Distal portal vende bir balık ağzı kesi olun. Forseps ile kesi açın ve portal ven cannulate. Karaciğer rengini yitiren kadar 20 ml Ringer solüsyonu ile karaciğer yıkayın ve bitişik harfler (ipek 6-0) ile kanül düzeltin. </li></ol></li><li> Arteriyel Kanülasyon (Kullanım bölüm 1.3 hazırlanan kanül) <ol><li> Çölyak gövde üzerinde aorta parçalara ayır. Retroperitoneal uzaydan aort segmenti Özgürleştirin. Aort yama oluşturmak için uzunlamasına aort segmenti kesin. Statik tutucu içine kendini inşa kanül yerleştirin. </li><li> Kanül üzerinde aort yama kayma 2 forseps kullanın. Kanül üzerinde ipek 6-0 ile hepatik arter bağlanır ve dayanak olarak yama sabitleyin. </li></ol></li><li> Karaciğer eksplantasyonu <ol><li> , Diyaframı açıp dairesel kesi yapmak ve çevre yapılardan karaciğer teşrih. </li></ol></li><li> Depolama <ol><li> Stokullanılana kadar 350 ml Ringer çözeltisi ile doldurulmuş, steril 500 ml tankında karaciğer yeniden. </li></ol></li></ol> 2. Deterjan Çözümleri <ol><li> % 10 Triton X-100 stok solüsyonu <ol><li> 900 ml su dest ile 1.000 ml&#39;lik şişe doldurun. ve 100 ml Triton X-100 (% 99,9) ekleyin. Triton X-100 eritmek için bir karıştırıcıyı kullanarak. Damıtılmıs şişeyi doldurun. 1000 ml&#39;lik toplam hacme kadar ulaşılır. </li></ol></li><li> % 1 Triton X-100 <ol><li> 1.000 ml şişe stok çözeltisi, 100 ml (% 10) ilave edilir. 900 mi su Hedef ekle. ve iki kez şişeyi çalkalayın. Steril bir filtre içerisinden,% 1 Triton X-100 çözeltisi filtre. </li></ol></li><li> % 10 SDS Stok Çözeltisi <ol><li> 900 ml su dest ile 1.000 ml&#39;lik şişe doldurun. ve 66.99 g SDS (% 99.9, yoğunluk 0.67) ekleyin. SDS çözmek için bir karıştırıcı kullanın. Damıtılmıs şişeyi doldurun. 1000 ml&#39;lik toplam hacme kadar ulaşılır. </li></ol></li><li> % 1 SDS <ol><li> 1.000 mi bot stok çözeltisi, 100 ml (% 10) ilave edilirtle. 900 mi su Hedef ekle. ve iki kez şişeyi çalkalayın. Steril bir filtre ile% 1 SDS çözeltisi filtre. </li></ol></li></ol> 3. Decellularization <ol><li> Perfüzyon Cihaz Set-up <img alt="Figür 1" src="/files/ftp_upload/53029/53029fig1.jpg" /></li></ol> Şekil 1:. Oscillating Basınç Koşullarda Seçici Arter ve Dört Rat karaciğerinin Portal Venöz Perfüzyon için Perfüzyon Cihaz Programı (. Struecker ark değiştirilmiş 14) <ol><li> Üç yollu ve dolum seviyesini düzenleyen ve 500 ml% 0,9 NaCl ile reaktörü doldurmak için Heidelberger uzantısı drenaj bağlayın. <img alt="Şekil 2," src="/files/ftp_upload/53029/53029fig2.jpg" /></li></ol> Şekil 2:. (4) portal venöz yoldan kabarcık kapanı (5) (2) ya da arteriyel ac yoluyla uzak ucuna perfüzyon set-up Plan perfüzyon odasına bağlantı (1.400 cm 3) (1)ri (3). Kabarcık kapanı (5) uzak ucunda bir tıkama (4) ve bir delik (6) ile donatılmış olmalıdır. Bir Heidelberger uzatma (7) kabarcık tuzak (5) bağlayın. Pompa segmenti (8) ile Heidelberger uzantısı bağlayın. Bundan başka, bir başka Heidelberger uzantısı (9), pompa segmenti (8) bağlanır. Deterjan çözeltisinin (10) şişenin içine geçen Heidelberger uzantısı (9) takın. (Ya da birden perfüzyonları durumunda basınç dağıtıcıya) perfüzyon odasına solunum cihazı (11) takın. Reaktör sıvıları boşaltmak için, atık şişe (12) çıkışı bağlayın. <ol><li> Pompanın içine pompa segmenti yerleştirin. PBS ile döngüsü doldurmak için pompayı çalıştırın, kabarcık kapanı distal ucunu kapatın ve havalandırma açın. Kabarcık alt güvenli PBS (2-3 cm) ile kaplı olduğunda, havalandırma kapatın ve baloncuk kapanı distal ucunu açın. </li><li> Karaciğer Kurulum <ol><li> İlgili reaktör erişmek için portal ven kanül üç yönlü stopcock bağlayın. Carteriyel erişim arteriyel kanül taşıyıcılarına bağlan- ması. Hava sistemlerini girmemesini sağlamak için dikkatli olun. </li></ol></li><li> Titreşimli Basınç Koşullar Uygulaması <ol><li> 15 bpm bir frekans ve 26 mbar (dak. 4 mbar, maks. 30 mbar), bir genlik solunum cihazı kullanın. Basınç dağılımı odasına solunum bağlayın ve reaktöre odasına bağlayın. Basınç uygulamasını başlatın. </li></ol></li><li> Decellularization Protokolü <ol><li> / Dakika, 5 ml bir akış oranı ile perfüzyon adımları. 90 dakika boyunca,% 1 Triton X-100 ile birlikte karaciğer perfüzyonu ile decellularization başlatın. Perfüzyon odasının sabiti içinde ve perfüze karaciğer üzerindeki sıvı seviyesini korumak için perfüzyon cihazının çıkışı yoluyla atık effluence izin verin. 90 dakika sonra,% 1 SDS deterjan değiştirin. <img alt="Şekil 3," src="/files/ftp_upload/53029/53029fig3.jpg" /> <img alt="Tablo 1" src="/files/ftp_upload/53029/53029table1.jpg" /></li></ol></li></ol> &lt;p&gt; Şekil 3: Tüm Deneysel Grupları için Perfüzyon Protokolü.

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The authors have nothing to disclose.

Sıçan karaciğer hasat ve decellularization sunulan tekniği kolayca tekrarlanabilir olmasına rağmen, dikkate bazı kritik adımlar vardır: Kan pıhtılaşmasını aktive edecek ve karaciğer içinde kan pıhtısı oluşumuna neden olabilir, çünkü karaciğer hasat için hazırlık sırasında, şiddetli kanama önlemek için önemlidir. Bizce, bu portal ven yoluyla perfüzyon sırasında hepatik arter yoluyla kan akışını önlemek için portal ven kanülasyonu önce doğrudan abdo...

Decellularization ve Recellularization vitro 1&#39;de işlevsel ve çoklu organ üretilmesini sağlayabilir. Kaldırma hücreleri ile antijenik madde ile (ör., DNA, a-Gal epitopları), bir organdan, olmayan ya da daha az immünojenik hücre dışı matris (ECM) elde edilebilir. Bu matris, bir organ, üç boyutlu mikroanatomisinin korur ve farklı, muhtemelen ksenogeneik kökenli 2 hücreleri ile yeniden popüle için ideal bir biyolojik matris olarak görev yapabilir. Bu durumda, bir hücresizleştirilmiş sıçan karaciğer matrisi insan karaciğer hücreleri ile yeniden doldurulur edilebilir. Bu insanlaştırılmış mikro-karaciğer hastalıkları (örn., Doğumsal metabolik hastalıklar, viral hastalıklar veya maligniteler) veya preklinik ilaç testi 3 araştırma için bir ex vivo modeli olarak hizmet verebilir. Sıçan karaciğer perfüzyon decellularization için çeşitli protokoller zaten 4-13 yayınlanmıştır. Tüm protokollerde, decellularleştirilmesi kanüllü portal ven aracılığıyla alkali iyonik veya non-iyonik deterjanlar perfüzyon ile elde edilmiştir. Bizim bilgimize göre, biz portal ven ve / veya sıçan hepatik arter 14 üzerinden seçici perfüzyon ile sıçan karaciğer decellularization bildiren ilk gruptu. Karaciğer farklı vasküler sistemlerinin seçici perfüzyon etkinleştirilmesi hücre yeniden popüle önemli bir rol oynayabilir, bundan başka, daha decellularization sonuçlar etkinleştirmek olabilir. Burada ayrıntılı çalışmada, karaciğer titreşen basınç koşullarında perfüzyon sağlayan bir ısmarlama tescilli perfüzyon cihazında perfüze edildi. Bu basınç koşulları karaciğer fizyolojik solunum bağımlı perfüzyon taklit: in situ, karaciğer hareketi solunum sırasında karaciğer perfüzyon üzerinde doğrudan etkisi vardır diyaframın copula altında asılı. İlham, özellikle optimize diyaframın düşürülmesi ve karaciğerde sıkma yol açarhepato-venöz çıkış, oysa son karaciğer yükselmesi ve portal venöz girişi 15 optimize karın içi basıncının düşürülmesi yol açar. Amacımız salınan basınç koşulları karın içi koşullar ex vivo taklit ederek sıçan karaciğer perfüzyon decellularization homojenliği üzerinde bir etkisi olup olmadığını araştırmaktır. decellularization sürecinin homojenliği perfüzyon decellularization bir hafife bir faktör olabilir. Bilinen tüm maddeler ECM karaciğer decellularization neden değişiklikler için kullanılır. Diğer alanlarda zaten tamamen decellularized oysa kötü perfüze alanlarda hücreler, ECM içinde kalır. Geri kalan hücrelerin çözmek için, perfüzyon süresi ya da basınç iyi perfüze alanlara daha fazla değişiklik neden yükselmiş olması gerekir. Bu nedenle, decellularization deterjanlar organ içindeki homojen dağıtılmalıdır.

<table><tbody><tr><td colspan="4">Self built arterial cannula</td></tr><tr><td>&amp;nbsp;Portex Non Sterile Polyethene Tubing</td><td>SIMS Portex</td><td>REF 800/110/100</td><td>0.28 mm ID 0.61 mm OD</td></tr><tr><td>&amp;nbsp;Portex Non Sterile Polyethene Tubing</td><td>SIMS Portex</td><td>REF 800/110/200</td><td>0.58 mm ID 0.96 mm OD</td></tr><tr><td>Venodrop Safe butterfly catheter</td><td>Fresenius Kabi</td><td>3275851</td><td>21 G</td></tr><tr><td colspan="4">portal vein cannula</td></tr><tr><td>Periphereal Venous Catheter</td><td>BD</td><td>393224</td><td>BD Venflon Pro 20G</td></tr><tr><td>Three-way stopcock</td><td>smiths medical</td><td>888-101RE</td><td></td></tr><tr><td colspan="4">surgery</td></tr><tr><td>Cotton Sticks</td><td>Hecht-Assistent</td><td>4302</td><td></td></tr><tr><td>Cotton Pads&amp;nbsp;</td><td>Shaoxing Zhengde Surgical dressing</td><td>13H118-03</td><td></td></tr><tr><td>Gauze Bandage</td><td>Hubei Haige&amp;nbsp; Medical Instruments</td><td>14388</td><td></td></tr><tr><td>&amp;nbsp;Ringer Solution</td><td>Fresenius Kabi</td><td>13 HKP022</td><td>1000 ml</td></tr><tr><td>10 ml Syringe</td><td>Braun</td><td>4606108V</td><td>10 ml/ Luer Solo</td></tr><tr><td>5 ml Syringe</td><td>Braun</td><td>4606061V</td><td>5 ml /Luer Solo</td></tr><tr><td>Suture (Silk 6/0)</td><td>Resorba</td><td>H1F</td><td>LOT 105001.81</td></tr><tr><td>medical drape</td><td>Shaoxing Zhengde Surgical dressing</td><td>D0613011</td><td></td></tr><tr><td colspan="4">surgical instruments</td></tr><tr><td>needle holder</td><td>Geuder</td><td>17570</td><td></td></tr><tr><td>micro-forceps</td><td>Inox-Electronic</td><td>91150-20</td><td></td></tr><tr><td>micro-scissors</td><td>Martin</td><td>11-740-11</td><td></td></tr><tr><td>micro-forceps</td><td>S&amp;amp;T&amp;nbsp;</td><td>112314</td><td></td></tr><tr><td>Clamp</td><td>Aesculap</td><td>BH111R</td><td></td></tr><tr><td>scissors</td><td>F S T&amp;nbsp;</td><td>14501-14</td><td></td></tr><tr><td>surgical forceps</td><td>Aesculap</td><td>BD 557</td><td></td></tr><tr><td colspan="4">Decellularisation</td></tr><tr><td>Respirator</td><td>Resmed</td><td>14.24.11.0004</td><td>SmartAIR ST</td></tr><tr><td>Perfusion Device</td><td>Charite, medical engineering laboratory</td><td>custome-made device</td><td>decellularisation device</td></tr><tr><td>peristaltic pump ismatec reglo ICC</td><td>IDEX</td><td>ISM4408&amp;nbsp;</td><td>4-channel</td></tr><tr><td>heidelberger extension 75 cm&amp;nbsp;</td><td>Fresenius Kabi</td><td>2873</td><td>75 cm</td></tr><tr><td>MS/CA pump-segment</td><td>IDEX</td><td>IS 3510&amp;nbsp;</td><td>MS/CA/click&#039;n&#039;go/POM-C</td></tr><tr><td>CA 2-stopper tube</td><td>Pharmed</td><td>BPT NSF-51</td><td></td></tr><tr><td>bubble trap&amp;nbsp;</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 colspan="4">Detergents</td></tr><tr><td>SDS pellets</td><td>Carl Roth&amp;nbsp;</td><td>CN30.4&amp;nbsp;</td><td>2.5 kg</td></tr><tr><td>Triton X-100</td><td>Carl Roth&amp;nbsp;</td><td>3051.1&amp;nbsp;</td><td>10 L</td></tr><tr><td>PBS&amp;nbsp;</td><td>Gibco</td><td>14190-094</td><td>DPBS</td></tr><tr><td colspan="4">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&amp;nbsp;</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.

homojenliği ve dolayısıyla farklı decellularization protokolleri etkinliği makroskopik gözleminden, histolojik analiz ve hücresizleştirilmiş karaciğer matrisleri içinde kalan DNA içeriğinin analizi ile değerlendirilmiştir. Ayrıca, korozyon döküm decellularization sonra karaciğerinin sağlam mikroanatomisinin görselleştirmek için yapıldı. Makroskopi Decellularization sırasında, karaciğer hücre içeriğine çıkarılmasını gösteren lu...

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.

Bir Salınım basınçlı perfüzyon Aygıt Sıçan karaciğerinin decellularization Prosedürü

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

procedure-for-decellularization-rat-livers-an-oscillating-pressure

Research

JoVE Journal

Bioengineering

10.2K Görüntüleme.  Charité – Universitätsmedizin Berlin. 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.

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

Decellularization and Recellularization of Whole Livers

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 recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

Tüm karaciğerleri Decellularization ve Recellularization

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 ...

Biyomühendislik

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

Subnormothermic Tekniği<em&gt; Ex vivo</em&gt; Karaciğer Depolama, Değerlendirme Perfüzyon ve Marjinal Karaciğer Greft Onarımı

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

Tıp

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing modified cannulas and techniques adapted from conventional commercial ex vivo perfusion equipment. The system was utilized to evaluate the preservation outcomes following 12 h of perfusion. C57BL/6J mice served as liver donors, and the livers were explanted by cannulating the portal vein (PV) and bile duct (BD), and subsequently flushing the organ with warm (37 &#176;C) heparinized saline. Then, the explanted livers were transferred to the perfusion chamber and subjected to normothermic oxygenated machine perfusion (NEVLP). Inlet and outlet perfusate samples were collected at 3 h intervals for perfusate analysis. Upon completion of the perfusion, liver samples were obtained for histological analysis, with morphological integrity assessed using modified Suzuki-Score through Hematoxylin-Eosin (HE) staining. The optimization experiments yielded the following findings: (1) mice weighing over 30 g were deemed more suitable for the experiment due to the larger size of their bile duct (BD). (2) a 2 Fr (outer diameter = 0.66 mm) polyurethane cannula was better suited for cannulating the portal vein (PV) when compared to a polypropylene cannula. This was attributed to the polyurethane material&#39;s enhanced grip, resulting in reduced catheter slippage during the transfer from the body to the organ chamber. (3) for cannulation of the bile duct (BD), a 1 Fr (outer diameter = 0.33 mm) polyurethane cannula was found to be more effective compared to the polypropylene UT - 03 (outer diameter = 0.30 mm) cannula. With this optimized protocol, mouse livers were successfully preserved for a duration of 12 h without significant impact on the histological structure. Hematoxylin-Eosin (HE) staining revealed a well-preserved morphological architecture of the liver, characterized by predominantly viable hepatocytes with clearly visible nuclei and mild dilation of hepatic sinusoids.

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

A normothermic ex vivo liver perfusion (NEVLP) system was created for mouse livers. This system requires experience in microsurgery but allows for reproducible perfusion results. The ability to utilize mouse livers facilitates the investigation of molecular pathways to identify novel perfusate additives and enables the execution of experiments focused on organ repair.

Normothermic Ex Vivo Karaciğer Makinesi Farede Perfüzyon

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing ...

Mühendislik

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Sıçan Vivo içinde kısmi karaciğer Dairesi'nde için bir temel olarak yeni bir cerrahi teknik

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Sıçan Portal Fibroblastlar izolasyonu ile<em&gt; Yerinde</em&gt; Karaciğer Perfüzyon

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Biyoloji

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Sıçan Karaciğer Hücreleri İzolasyon ve İlköğretim Kültür

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

Vaskülarize Kompozit Allotransplantasyon için Ex Vivo Perfüzyon Bazlı Biyoreaktör Kullanılarak Sıçan Arka Bacaklarının Tedariği ve Desellülarizasyonu

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

Ex Vivo Normothermic karaciğer perfüzyon küçük bir hayvan Model

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Primer Sıçan Hepatositlerinin Çok Parametreli Perfüzyon Kontrolü ile İzolasyonu

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

Evaluation of Apoptosis and Lung Injury in DCD Donor Lungs Preserved by EVLP

Establishment of a Novel Ex Vivo Lung Perfusion System for Rat Lungs After Circulatory Death

The increasing scarcity of donor lungs for transplantation has spurred significant interest in the utilization of organs procured from donors after cardiac death (DCD). However, a critical challenge remains in maximizing the number of viable lungs that meet stringent criteria for clinical use. Ex vivo lung perfusion (EVLP) emerges as a promising technology to evaluate and potentially improve the function of donated lungs. This paper describes the development of a cost-effective and reproducible small animal experimental platform specifically designed for EVLP research. This platform offers a high degree of customization, enabling researchers to tailor experimental parameters to address specific research questions within the field of EVLP. The team established a rat model to simulate DCD and implemented a two-stage preservation approach. Lungs were subjected to static cold storage using a perfusate solution at 4 &#176;C for 4 h, followed by normothermic EVLP for another 4 h. Researchers evaluated various lung function parameters, including pulmonary vascular resistance, pulmonary dynamic compliance, blood gas measurements of the perfusate, and apoptotic cell death in lung tissues. The study demonstrates that EVLP could significantly improve the functional performance of DCD lungs compared to simple cold storage at 4 &#176;C, suggesting that EVLP has the potential to mitigate ischemia-reperfusion injury and enhance graft function following circulatory death, thereby increasing the pool of transplantable lungs.

The objective is to develop a new ex vivo lung perfusion (EVLP) model in rats that can sustain consistent lung function for 4 h throughout EVLP and furnish the industry with implementation specifics that will offer a sturdy method for applying EVLP to rodent models.

The increasing scarcity of donor lungs for transplantation has spurred significant interest in the utilization of organs procured from donors after ...

Bir Salınım basınçlı perfüzyon Aygıt Sıçan karaciğerinin decellularization Prosedürü

Özet

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