Bu çalışma araştırma için insan dokuları kullanılabilir hale İnsan Doku ve Hücre Araştırma Vakfı tarafından mümkün olmuştur. Ve Hepacult GmbH bu iş için finansal destek Federal Eğitim ve Araştırma Bakanlığı (: Sanal Karaciğer Ağ, hibe sayısı 0.315.759 hibe adı) alındı. Bizim sayesinde de karaciğer perfüzyon ve hepatosit izolasyonu yapılması için karaciğer örnekleri ve Hücre İzolasyon Çekirdek Tesisinden teknik asistanların koleksiyonu için Grosshadern Hastanesi Doku bankası teknik asistanlar gidin. Özellikle, biz video Bu prosedürü göstermek için Natalja Löwen teşekkür etmek istiyorum. Son olarak, videonun şematik genel Şekil 1 için illüstrasyonlar ve şekiller oluşturma için Natalja Löwen ve Edeltraud Hanesch teşekkür etmek istiyorum.

1.. Perfüzyon ve Yalıtım Çözümleri hazırlanması <ol><li> Karaciğer parçası ve Tablo 1 &#39;e göre hepatositlerin izolasyon perfüzyon için gerekli olan çözümler hazırlayın. Çözümler, kullanılana kadar 4 ° C&#39;de muhafaza edilebilir. </li><li> Steril filtre 0.22 mikron filtre kullanılarak tüm çözümler. </li><li> Karaciğer ile temas eden bütün çözeltiler steril olmalıdır. </li></ol> 2. Perfüzyon Ekipman ve Çözümleri hazırlanması <ol><li> Şekil 1 &#39;de gösterildiği gibi, karaciğer parçanın perfüzyon için donatım kadar ayarlanmalıdır. </li><li> Su banyosu her bir deneysel kurulum farklıdır uygun bir sıcaklıkta, en ayarlanmalıdır da karaciğer parça ulaştıklarında çözeltiler 37 ° C sıcaklıkta şekildedir. Bu durumda, su banyosu Çözümleri 1, 2 ve 3 ile kaplanmış cam soğutucu ısınmaya 41 ° C&#39;de ayarlanır. Çözüm 4 ısındı edilmelidirKollajenaz aktivite kaybını azaltmak için kullanım için ayrı bir su banyosu içinde 37 ° C. </li><li> Kısa bir süre karaciğer perfüzyon önce,% 95 O 2 /% 5 CO 2 oksijenlenme aparatı (Şekil 1E) gaz içeren gaz tankı regülatör açın. </li></ol> 3. Karaciğer Perfüzyon <ol><li> Sadece 1 kesme yüzeyi ile ideal mümkün ve olduğunca sağlam Glisson kapsülü ile bir karaciğer parçası perfüzyon için bir patolog elde edilmelidir. </li><li> Delikli bir filtre diski (Şekil 1 B) içeren bir Büchner hunisi üzerinde, bu parça karaciğer yerleştirin. </li><li> Perfüzyon sistemi Çözüm 1 ile astarlanmalıdır. </li><li> Düşük bir akış oranı ile, zeytin uçları eğri bir sulama kanüller karaciğer parçasının kesme yüzeyi üzerinde daha büyük kan damarlarının içine yerleştirilmelidir. Kan karaciğerden dışarı basması gibi, doku perfüzyonu iyi olan yerlerde hafif olur. Çeşitli boyutlarda için kullanılan kanül sayısıkaraciğerinin Şekil 2A&#39;da gösterilmiştir. Seçilen göstergesi boyutu yerinde kanüller yapacak oturması sonuçlanmalıdır. Küçük kan damarları, karaciğer parçasının dışarı boşaltmak için perfüzyon tamponu için açık bırakılmalıdır. </li><li> Karaciğer (Şekil 2B) büyüklüğüne bağlı olarak, ml / dak 110-460 arasına peristaltik pompa ile ilgili akış hızını artırır. Bu, 44 ± 16 arasında bir ortalama akış oranında ml / dak kanül başına (Şekil 2C) ile sonuçlanır. Seçilen hızı karaciğer parça bağlıdır ve karaciğer parçasının hafif plumping kadar sonuçlanmalıdır. Bazı durumlarda, yukarıda bahsedilen hafif plumping elde etmek için mikro vasküler kıskaçlar ile açık kapların bir kapalı sıkıştırılması için gerekli olabilir. Karaciğer parçası boyunca açık renk olduğunda iyi bir perfüzyon görülebilir. </li><li> Fizyolojik ile ıslatılmış gazlı bez parçası ile kaplayıp perfüzyon sırasında nemli karaciğer parçasını tutun. </li><li> Herhangi r dışarı atılması için Çözüm 1 1 L serpmekkaraciğer parça kan emaining. </li><li> Çözüm 2 perfüzyon sıvısı değiştirin ve 10 dakika boyunca perfüze. </li><li> Çözüm 3 perfüzyon sıvısı açın ve 0,5 L. ile serpmek </li><li> Kollajenaz (Tablo 2) 0.1-0.15% içerir Çözüm 4&#39;e perfüzyon sıvısını değiştirin. </li><li> Karaciğer yeterli sindirilir kadar bu adım için, perfüzyon 9-12 dakika boyunca, bir devri daim şekilde gerçekleştirilebilir veya olmalıdır; karaciğer dokusu Glisson kapsülü altında ayrı hafif kırmak ve bir de kör tarafı ile problanmıştır zaman yumuşatılmış hissetmek görünmelidir neşter. </li></ol> 4. Hepatositlerin izolasyonu <ol><li> Peristaltik pompayı kapatın ve karaciğer parça kanüller kaldırın. </li><li> Çözüm 5 100-200 ml ihtiva eden bir kristalizasyon çanağında karaciğer parça yerleştirin. </li><li> Dikkatle Glisson kapsülü kaldırın ve yavaşça hücreleri silkelemek. De perfüze olmayan bölgeler yoksa, bir neşter kesmek için kullanılabilirBu bölgeler içinde bulunan hücreleri serbest bırakmak için. Gibi sürecinde ihtiyaç duyulan daha Çözüm 5 ekleyin. </li><li> 500 ml&#39;lik bir nihai hacim elde edilene kadar daha fazla Çözüm 5 ekleyin. </li><li> Iki hücre süspansiyonu filtre, ilk önce bir 70 mikron naylon elek, ardından bir 210 um naylon elekle. Daha sonra, 200 ml santrifüj tüplerine hücre süspansiyonu dökün. </li><li> 4 ° C&#39;de 5 dakika boyunca 72 g hücre süspansiyonu santrifüj Aspire edilen süpernatan ve yavaşça Çözüm 5 200 ml yavaşça tekrar süspansiyon hücre topağı. </li><li> Yıkama adım numarasını 4.6 üç kez tekrarlayın. Son santrifüj adım, soğuk depolama çözümü tekrar süspansiyon hücreleri (malzemelerin listesine bakınız). Hücreler, bir hemositometre tabanlı bir tripan mavi dışlama tahlili kullanılarak verim ve canlılığı değerlendirilmesi için mililitre başına yaklaşık 2-5000000 hepatositler olmalıdır. </li><li> Bir tripan mavi dışlama tahlili gerçekleştirmek için, tripan mavi soluti, 0.5 ml ihtiva eden bir mikrofüj tüpüne (≈ 2-5000000 / ml), uygun şekilde seyreltilmiş 0.1 ml hücre eklemekon (% 0.4 tripan mavi fosfat tamponlu tuzlu su (PBS) içinde çözülmüş) ve 0.4 ml PBS. İyice karıştırdıktan sonra hücre süspansiyonu, süspansiyon ile bir hemasitometre yük ve 100X büyütme ile mikroskop altında incelenmiştir. Canlı hücreler boyanmamış görünür iken mikroskobu altında, ölü hücreleri mavi boyanmış olacaktır. Menositometrede işaretli 1 mm 2 bloğun her birinde canlı ve toplam hücre sayısını. Canlılık (%), canlı hücre verimi (ml soğuk depolama çözümü (CSS) başına milyon hepatositler veya g karaciğer başına milyon hepatositler) aşağıdaki formüller kullanılarak hesaplanabilir. </li></ol><img alt="Denklem 1" fo:src="/files/ftp_upload/50615/50615eq1highres.jpg" src="/files/ftp_upload/50615/50615eq1.jpg" /> Not: Bu durumda, tripan mavi seyreltme faktörü 10 ve hemositometre faktör 10.000 &#39;dir.

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Bu protokolün optimizasyonu kısmen Hepacult GmbH bir hibe ile finanse edildi. Dr Wolfgang THASLER Hepacult GmbH kurucularından biri olan ve bu şirkette yönetim kurulu üyelerinden biri olmaya devam etmektedir. Maresa Demmel istihdam Hepacult tarafından kısmen. Maria Hauner Hepacult GmbH tarafından istihdam edilmektedir. Hepacult rızası ve araştırma amaçlı açık erişimli insan hepatositleri sunmaktadır Üniversitesi&#39;nden bir spin-off biyoteknolojik firmadır.

Bu protokol, yüksek sağlamlık ve saflıkta insan hepatositlerinin izolasyonu ile sonuçlanır. Bu sonuçları elde etmek için, bu karaciğer uygun bir parça ile başlamak önemlidir. Karaciğer parçası 1 kesme yüzeyi hariç tüm yüzeylerde bozulmadan Glisson kapsülü olmalıdır. Bir diğer önemli faktör farklı partisi sindirim 11 sonra hepatositlerin canlılığında belirgin farklılıklara neden olabilir olarak, kullanılan kolajenazın belirli bir küme olduğu. Bu nedenle, kolajenaz, farklı...

A correction was made to: <a href="http://www.jove.com/video/50615/isolation-human-hepatocytes-two-step-collagenase-perfusion">Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure </a>
The changes listed below have been made to Table 1.
1). In the recipe for Solution 2, the Final Concentration of EGTA has been changed from:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>EGTA&#160;</td>
			<td>0.1mM</td>
		</tr>
	</tbody>
</table>
to:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>EGTA&#160;</td>
			<td>1mM</td>
		</tr>
	</tbody>
</table>
2). In the recipes for Solution 3 and 4, the Final Concentration of Calcium chloride dihydrate has been changed from:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Calcium chloride dihydrate&#160;</td>
			<td>0.5&#181;M</td>
		</tr>
	</tbody>
</table>
to:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Calcium chloride dihydrate&#160;</td>
			<td>5mM</td>
		</tr>
	</tbody>
</table>

A correction was made to: Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure.
The Final Concentration of EGTA in Solution 2 and the Final Concentration of Calcium chloride dihydrate in solutions 3 and 4 have been updated in Table 1.

Erratum: Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

Bir karaciğer hücre süspansiyonu, mekanik ya da enzimatik yöntemlerle karaciğer hazırlanabilir. Bütün karaciğer hücreleri hazırlamak için kullanılan mekanik yöntemler, tülbentten 2 vasıtasıyla karaciğeri zorlayan bir Kahn çalkalayıcı 3 cam boncuklar ile bir karaciğer parçası sallayarak, yılda vb gevşek havan elleri 4,5 ile cam homohenizatörlerin kullanarak dahil, mekanik yöntemler dışına düşmüş nedeniyle hücre zarlarına hasar ve izole edilmiş hepatositlerin 6,7 fonksiyon kaybına lehine. Sonuç olarak, bir enzimatik yöntem şu anda kullanımı hepatositlerin izolasyonu için ana yöntem. Berry ve Friend 8 sıçanlarda portal ven aracılığıyla karaciğerden kolajenaz ve hiyalüronidaz perfüze zaman, bir enzimatik yöntem ile hepatositlerin izolasyonu büyük ölçüde geliştirilmiştir. Bu perfüzyon işlemi yol açan, enzimler, hücrelerin çoğunluğu ile yakın temas için izin vermek üzere damarsal kullanılanhepatositlerin 8 veriminde 6-katlık bir artış. Bundan başka, bu yöntem, hemen hemen izole vezikülleri içine endoplazmik retikulum hiçbir dönüşüm ve hiçbir zarar mitokondriyal 8, yapısal bütünlüğünü muhafaza hücreleri elde edildi. Bu yöntem, karaciğer hücre izolasyonu için iki aşamalı bir prosedür perfüzyon öncülük Seglen 1, modifiye edilmiştir. Bu prosedürde, sıçan karaciğer Ca 2 + 1 ihtiva eden bir kolajenaz perfüzyon tampon maddesi ile, ardından bir Ca2 + serbest tamponu ile perfüze edilmiştir. Ikinci aşamada Ca 2 + eklenmesinin uygun kolajenaz aktivitesi 1.9 için gerekli iken, birinci aşamada Ca 2 + kaldırılması, dezmozomlar bozmak için yardımcı olur. Yukarıda tarif edilen yayınlanmış çalışma sıçanlarda gerçekleştirildi olduğu göz önüne alındığında, bu makalede yüksek uğultu canlılığı ile hepatositlerin izolasyonu için kullanılabilecek modifiye edilmiş bir prosedürü ortaya koymayı amaçlamaktadırBir ciğeri. İnsan hepatositlerinin kullanılması translasyonel araştırma için ve hayvan modelleri kullanılarak deneyler doğrulamak için önemli olmaya devam etmektedir. Bu çalışmada kullanılan insan karaciğer parçaları İnsan Doku ve Hücre Araştırma Vakfı, bir devlet kontrolündeki kâr amacı gütmeyen vakfın 10 ile yönetişim için rıza ile elde edildi. Bir patolog tanı için gerekli ne uzaklaştırıldıktan sonra, karaciğer parçaları ve geri kalan dokulardan toplanmıştır. Patolog tarafından kapalı kesitli doku morfolojik karaciğer rezeksiyonu sonrası rezeksiyon marjları elde edilen sağlıklı doku oldu.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Bubble trap</td>
			<td>Ga&szlig;ner Glastechnik</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass jacketed condenser</td>
			<td>Ga&szlig;ner Glastechnik</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>41 &deg;C Water bath</td>
			<td>Julabo</td>
			<td>35723-H24/EG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>37 &deg;C Water bath</td>
			<td>GFL</td>
			<td>1083</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compressed gas cylinder (95 % O2/5 % CO2)</td>
			<td>Linde</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gas permeable tubing</td>
			<td>Neolab</td>
			<td>2-4440</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Ismatec</td>
			<td>IP65</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Feather</td>
			<td>320010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Omnilab</td>
			<td>5171014</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Conical flasks 1 L</td>
			<td>Schott Duran</td>
			<td>2121654</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Conical flasks 5 L</td>
			<td>Schott Duran</td>
			<td>2121673</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Beakers</td>
			<td>Schott Duran</td>
			<td>2110654</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>200 ml centrifuge tubes</td>
			<td>Becton Dickinson</td>
			<td>352075</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Crystallizing dish</td>
			<td>Omnilab</td>
			<td>5144063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Curved irrigation cannulae with ball tips</td>
			<td>Ernst Kratz GmbH</td>
			<td>1464LL/ 1465LL A+B/ 1472LL</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro vascular clamps</td>
			<td>Ernst Kratz GmbH</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B&uuml;chner funnel</td>
			<td>Carl Roth</td>
			<td>HT38.1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nylon mesh 210 &mu;m</td>
			<td>Neolab</td>
			<td>4-1413</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nylon mesh 70 &mu;m</td>
			<td>Neolab</td>
			<td>4-1419</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.22 &mu;m sterile filters</td>
			<td>Peske</td>
			<td>99505</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>500 ml bottles</td>
			<td>Schott Duran</td>
			<td>2180144</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 L bottles</td>
			<td>Schott Duran</td>
			<td>2180154</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Peske</td>
			<td>06-0001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1.5 ml tubes</td>
			<td>Eppendorf</td>
			<td>0030 120,086</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>BD Biosciences</td>
			<td>352070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td>Neolab</td>
			<td>1508454</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile Pasteur pipettes</td>
			<td>Brand</td>
			<td>747715</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Motorised pipette filler (Pipette boy acu)</td>
			<td>Integra</td>
			<td>155017</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Refridgerated centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laminar flow</td>
			<td>Kendro</td>
			<td>Hera safe-KS9</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aspirator (Low-flow surgical suction pump)</td>
			<td>Atmos</td>
			<td>C361</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laboratory Gas Burner</td>
			<td>Integra</td>
			<td>Fire Boy eco</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Disposable laboratory coat</td>
			<td>Paperlynen GmbH</td>
			<td>MD0202414</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical mask with visor</td>
			<td>Kimberly-Clark</td>
			<td>48247</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical hood</td>
			<td>Barrier</td>
			<td>42072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Latex gloves</td>
			<td>Semper Care</td>
			<td>CE0321</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase (Batch number NB 4G)</td>
			<td>Serva</td>
			<td>17465</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Merck</td>
			<td>2382</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Roth</td>
			<td>9265.2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Roth</td>
			<td>9105.3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Serva</td>
			<td>26868</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>Biomol</td>
			<td>01400-2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Serva</td>
			<td>22700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate</td>
			<td>Serva</td>
			<td>30180</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.4% Trypan blue solution</td>
			<td>Lonza</td>
			<td>17-942E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cold storage solution</td>
			<td>Hepacult GmbH</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

isolation of human hepatocytes by a two-step collagenase perfusion procedure

Karaciğer, istisnai bir rejenerasyon kapasitesine sahip bir organ, örneğin detoks, metabolizma ve homeostasis gibi işlevleri geniş bir yelpazede, yürütmektedir. Gibi, hepatositler araştırma soruları büyük bir çeşitlilik için önemli bir model vardır. Özellikle, insan hepatosit kullanımı farmakokinetik, toksikoloji, karaciğer rejenerasyonu ve translasyonel araştırma alanlarında özellikle önemlidir. Bu yüzden, bu yöntem, Seglen 1 tarafından tarif edildiği gibi hepatositleri izole etmek için iki aşamalı bir kolajenaz perfüzyon prosedürünün modifiye edilmiş bir versiyonunu göstermektedir. Daha önce, hepatositler, mekanik yöntemlerle izole edilmiştir. Bununla birlikte, enzimatik yöntemler hepatosit izolasyonundan sonra, yapısal bütünlüğü ve işlevi muhafaza üstün olduğu gösterilmiştir. Burada yer Bu yöntem, 77 ±% 10 arasında bir canlılığı ile hepatosit büyük bir verimle insan karaciğer parçaları ve sonuçlar sıçan karaciğer için daha önce tasarlanmış yöntemini uyarlar. AnaBu prosedürde fark kan damarlarının kanülasyon işlemidir. Bundan başka, burada tarif edilen yöntem, ayrıca, karaciğer karşılaştırılabilir veya kan damarı boyutları ile diğer türlerden elde edilen karaciğer uygulanabilir.

Perfüzyon Kur Karaciğer perfüzyon için gerekli ekipman, Şekil 1&#39;e göre ayarlanmış olmalıdır. Canlılık ve İzole İnsan Hepatositlerin Verim Izole edilmiş insan hepatositlerinin ortalama canlılığı, 77 ±% 10 ve hepatositlerin ortalama verim ± standart sapma olarak ifade edilen değerler ile, 13 ± 11000000 hepatositler / g karaciğer idi. Hepatosit izolasyonların sayısı bu ortalama 648 izolasyonları...

Watch this Scientific Journal Video about Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure at JoVE.com

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

İnsan hepatositlerinin izole edilmesi için bir modifiye edilmiş iki aşamalı bir kolajenaz perfüzyon prosedür tarif edilmektedir. Bu yöntem, aynı zamanda, diğer memeli karaciğerinden uygulanabilir. Izole hepatositler onları böyle karaciğer rejenerasyonu, farmakokinetik ve toksikoloji gibi alanlarda bilimsel araştırmalar için uygun bir model yapma, yüksek verim ve canlılığı mevcuttur.

Bir iki aşamalı kolajenes perfüzyon prosedür ile insan Hepatositlerin İzolasyonu

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

isolation-human-hepatocytes-two-step-collagenase-perfusion

Research

JoVE Journal

Biology

30.8K Görüntüleme.  Grosshadern Hospital, Munich.  İnsan hepatositlerinin izole edilmesi için bir modifiye edilmiş iki aşamalı bir kolajenaz perfüzyon prosedür tarif edilmektedir. Bu yöntem, aynı zamanda, diğer memeli karaciğerinden uygulanabilir. Izole hepatositler onları böyle karaciğer rejenerasyonu, farmakokinetik ve toksikoloji gibi alanlarda bilimsel araştırmalar için uygun bir model yapma, yüksek verim ve canlılığı mevcuttur. 

Video: Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Immunosurgery İnsan Embriyonik Kök Hücreleri türetilmesi

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Biyoloji

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 ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

Langerhans İzolasyonu Fare Islet Saflaştırılmış Collagenase ve Nötr Proteaz bir kombinasyonu kullanılarak

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

GST-His saflaştırma: Tam boy Saflaştırılmış Proteinler Verim iki aşamalı bir saflaştırma protokolü Affinity

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

İncelenmesi<em&gt; Drosophila</emIşık Mikroskopi tarafından&gt; Larva Trakeal Terminal Hücreleri

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

İlköğretim İnsan Hepatositlerin İzolasyonu ve Non-parankimal karaciğer hücrelerinin Binbaşı popülasyonları İlgili Protokolü

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

İki adımlı PCR ve Yeni Nesil 16S rRNA Gen Sıralaması Kullanarak Mikrobiyota Analizi

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

Memeli Pluripotent Hücreleri Üretmek için Epigenetik Modifikasyonu ve Biyomekanik İpuçlarını Birleştiren İki Adımlı Bir Strateji

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

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 ...

İki aşamalı mitokondriyal izolasyon kullanılarak geliştirilmiş protein miktar tayini

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial proteome is composed of more than 1,000 proteins, and the abundance of each can vary dynamically in response to external stimuli or during disease progression. Here, we describe a protocol for isolating high-quality mitochondria from primary cells and tissues. The two-step procedure comprises (1) mechanical homogenization and differential centrifugation to isolate crude mitochondria, and (2) tag-free immune capture of mitochondria to isolate pure organelles and eliminate contaminants. Mitochondrial proteins from each purification stage are analyzed by quantitative mass spectrometry, and enrichment yields are calculated, allowing the discovery of novel mitochondrial proteins by subtractive proteomics. Our protocol provides a sensitive and comprehensive approach to studying mitochondrial content in cell lines, primary cells, and tissues.

Yazar Spotlight: Gelişmiş Protein Keşfi ve Miktar Tayini için Mitokondrinin İki Adımlı Etiketsiz İzolasyonu  

We present a two-step protocol for high-quality mitochondria isolation that is compatible with protein discovery and quantification at a proteome scale. Our protocol does not require genetic engineering and is thus suitable for studying mitochondria from any primary cells and tissues.