Biz bu projenin çeşitli aşamalarında yardım, tavsiye ve kritik girişi sağlanan bütün arkadaşlarımıza teşekkür etmek istiyorum. Bu video makalenin Üretim IMIDIA (fonlarla desteklenen <a href="http://www.imidia.org" target="_blank">http://www.imidia.org</a> ), Eğitim ve Diyabet Araştırma Alman Merkezi (DZD için Araştırma (BMBF) için Alman Bakanlığı <a href="http://www.dzd-ev.de" target="_blank">http://www.dzd -ev.de</a> ) ve Teknoloji Dresden Üniversitesi Üniversite Hastanesi Carl Gustav Carus. Bu yayın için önde gelen iş hibe anlaşması n ° 155005 (IMIDIA), Avrupa Birliği Yedinci Çerçeve Programı (FP7/2007-2013) ve EFPIA verilen mali katkı oluşmaktadır hangi kaynaklar altında Yenilikçi İlaçlar Girişimi Ortak Taahhüt destek aldı şirketlerin ayni katkılar.

1. İnsan Pankreas Doku Dondurucu <ol><li> Yağ dokusu, kan damarları, sinirler ve neşterin ve cımbız ile non-parankimal doku çıkarın ve parçaları (~ 0,5-1 cm&#39;lik küpler) içine pankreatik doku kesti. </li><li> Bir Cryomold merkezinde tek pankreas dokusu parça yerleştirin ve soğuk Tissue-Tek Ekim bileşiği ile tamamen kapsamaktadır. Kimin altında sıvı azot içinde önceden soğutulmuş 2-metilbutane ve ek dondurma ile kaplı bir kavanoza Cryomold koyun. -80 ° C&#39;de dondurulmuş numune depolamak </li></ol> 2. Dondurulmuş Bölümler hazırlanması <ol><li> RNA denatürasyon önlemek için tüm adımları boyunca eldiven giyin. </li><li> Dışarda soğuk% 100 etanol ve RNaz ile kriyostat bıçak, Numune tutucu ve fırça temizleyin. Kriyostat içindeki dondurulmuş doku yerleştirin. </li><li> Doku, bıçak ve fırça -20 ° C&#39;ye ulaşıncaya kadar bekleyin Beklerken kriyostat odası içinde SuperFrost Plus slaytlar ve pre-serin bunları etiketleyindoku -20 ° C&#39;ye ulaşması için </li><li> Numune tutucu üzerindeki Tissue-Tek Ekim Bileşik sürün ve üstüne dondurulmuş doku yerleştirin. </li><li> Tissue-Tek Ekim Bileşik dondurulur kez cryoblock Döşeme ve bir jilet ile Tissue-Tek Ekim Bileşik herhangi bir aşırı kaldırın. </li><li> Pankreatik doku bloğundan 40 ardışık 10 mikron dilim toplam kesin. Ayrıca biz mikrodiseksiyon sürecinde dilimleri içinde adacıklar belirlenmesini kolaylaştıracak pankreatik kısmın genel çizimler için kaydedilmek üzere ilk ve orta dilim kesmek tavsiye. </li><li> Bıçak bıçak bölümüne uyacaktır sadece bölgeyi ısıtmak için parmağınızı (bir eldiven ile kaplı) ile slayt arka dokunarak SuperFrost Plus slayt merkezine bölümleri aktarın. </li><li> -20 Kriyostat içindeki bölümlerde 1-2 dk ° C, kuru buz üzerinde yerleştirilen bir slayt kutunun içine koyun sonra kurulayın. Bölümlerini depolamak-80 ° C </li><li> Gerekirse, kağıt havlu ve soğuk% 100 etanol ile bıçak temizleyin. </li></ol> 3. Dondurulmuş Bölümler Dehidrasyon <ol><li> Reaktifler hazırlayın: 30 ml taze hazırlanmış% 70 etanol, 30 ml DEPC işlenmiş su, 2x30 ml% 100 etanol ve 50 ml Cellstar borular (Şahinleri) içine 30 ml ksilen dökmek, soğuk ve buz üzerinde tüm reaktifler (ksilen hariç) tutmak. </li><li> % 100 etanol ve RNaseZAP ile kaputun altında çalışma alanı temizleyin. </li><li> Şöyle Süreç iki cryosections: buz DEPC-işlenmiş su 5-6 hızlı dips ile yıkayın, 30 saniye boyunca buz gibi soğuk% 70 etanol içinde düzeltmek, ksilen 4 dakika inkübe edilir, buz% 100 etanol içinde 1 dakika boyunca iki kez kurutmak oda sıcaklığında (25 ° C), ve 3-5 dakika boyunca kuru hava at. Cryosections başka bir çift ile bu adımı yineleyin. </li><li> Işık ve nemden korumak için alüminyum folyo ile sarılmış bir 50 ml Cellstar tüp içine sırt sırta iki kurutulmuş cryosections yerleştirin. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. PALM MicroBeam, Zeiss ile Beta-hücreleri Lazer Diseksiyon <ol><li> RNaseZAP ile mikroskop temizleyin ve RoboMover yılında Yapıştırıcı Cap monte. </li><li> Filtre seti 09 (uyartım: 450-490 nm, emisyon&gt; 515nm), AutoLPC modu,% 50 lazer enerjisi,% 65 lazer odak,% 100 lazer hız, AutoLPC çekim arasında 5 mm mesafe, 6 mikron mesafe aşağıdaki ayarları belirleyin -10100: hattına Çalışma yüksekliği. </li><li> Aşamasında içine bir slayt yerleştirin. </li><li> Mikroskopik (5x veya 10x objektif) altında autofluorescent beta hücreleri bulun. </li><li> 40x lense geçiş, &quot;Freehand&quot; seçim aracı ile beta hücreleri seçin ve lazer kullanarak bunları microdissect. </li><li> Tek AdhesiveCap içine dört susuz cryosections dokusu yakalayın. 1 Yorum: Bir susuz cryosection ve beta hücreleri incelemek için zaman neden olacaktır doku bölümleri rehidrasyon önlemek için 10-20 dakikadan uzun olmamalıdırBozulması RNA. 2 Yorum: kapısına kadar sahne taşıdıktan sonra görsel inceleme ile başarılı mikrodisseksiyon işlemi doğrulayın. </li></ol> 5. Microdissected Beta Hücre Zenginleştirilmiş Doku parçalanması <ol><li> RoboMover dan AdhesiveCap çıkarın. </li><li> AdhesiveCap bir kapak ve 42 de ters inkübe ° C 30 dakika boyunca içine Pipet 10 ul ekstraksiyon tamponu (XB, PicoPure RNA İzolasyonu Takımı, Arcturus). </li><li> 10,000 xg&#39;de aşağı spin ve kuru buz üzerinde lizat koydu. RNA ekstraksiyonu yapılır kadar -80 ° C&#39;de saklayın. </li><li> Tekrar 3. adımları yineleyin.) 5.) Diğer tüm bölümler. </li></ol> 6. RNA Ekstraksiyon <ol><li> Tüm on 10 ul lizatları birleştirin </li><li> % 70 Etanol (DNaz Tedavi dahil) 1:1 oranında Ekstraksiyon Tamponu (XB) kullanarak, PicoPure RNA İzolasyonu Takımı, Arcturus protokole göre oda sıcaklığında çalışarak RNA izolasyonu ile devam edin. </li></ol> 7. RNA Analizi <ol><li> Bir Agilent 2100 Bioanalyser (picoChip&#39;in) kullanarak kalite ve miktar analizi için 1 ul RNA kullanın. Kantitatif değerlendirme çip üzerinde paralel olarak yüklü bir RNA için referans standart olarak yapılır. </li></ol>

<ol>
	<li>Bonner, R. F., Emmert-Buck, 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=Laser+capture+microdissection:+molecular+analysis+of+tissue.">Laser capture microdissection: molecular analysis of tissue.</a> Science. 278, 1481-1483 (1997).</li><li>Suarez-Quian, C. A., Goldstein, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+of+single+cells+from+complex+tissues.">Laser capture microdissection of single cells from complex tissues.</a> Biotechniques. 26 (2), 328-3235 (1999).</li><li>Espina, V., Wulfkuhle, J. D., 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=Laser-capture+microdissection.">Laser-capture microdissection.</a> Nature Protocols. 1, 586-603 (2006).</li><li>Mikulowska-Mennis, A., Taylor, T. 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=High-quality+RNA+from+cells+isolated+by+laser+capture+microdissection.">High-quality RNA from cells isolated by laser capture microdissection.</a> Biotechniques. 33 (1), 176-179 (2002).</li><li>B&#246;tticher, G., Sturm, D., Ehehalt, F., Knoch, K. P., Kersting, S., Gr&#252;tzmann, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Human+Islets+from+Partially+Pancreatectomized+Patients.">Isolation of Human Islets from Partially Pancreatectomized Patients.</a> J. Vis. Exp. (53), e2962 (2011).</li><li>Marselli, L., Sgroi, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+of+human+pancreatic+beta-cells+and+RNA+preparation+for+gene+expression+profiling.">Laser capture microdissection of human pancreatic beta-cells and RNA preparation for gene expression profiling.</a> Methods in Molecular Biology. 560, 87-98 (2009).</li><li>Marselli, L., Thorne, 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=Gene+Expression+of+Purified+{beta}-Cell+Tissue+Obtained+from+Human+Pancreas+with+Laser+Capture+Microdissection.">Gene Expression of Purified {beta}-Cell Tissue Obtained from Human Pancreas with Laser Capture Microdissection.</a> Journal of Clinical Endocrinology &amp; Metabolism. 93, 1046-1053 (2008).</li><li>Bottino, R., Balamurugan, A. N., 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=Response+of+human+islets+to+isolation+stress+and+the+effect+of+antioxidant+treatment.">Response of human islets to isolation stress and the effect of antioxidant treatment.</a> Diabetes. 53 (10), 2559-2568 (2004).</li><li>Abdelli, S., Ansite, 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=Intracellular+stress+signaling+pathways+activated+during+human+islet+preparation+and+following+acute+cytokine+exposure.">Intracellular stress signaling pathways activated during human islet preparation and following acute cytokine exposure.</a> Diabetes. 53 (11), 2815-2823 (2004).</li><li>Negi, S., Jetha, 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=Analysis+of+beta-cell+gene+expression+reveals+inflammatory+signaling+and+evidence+of+dedifferentiation+following+human+islet+isolation+and+culture.">Analysis of beta-cell gene expression reveals inflammatory signaling and evidence of dedifferentiation following human islet isolation and culture.</a> PLoS One. 7 (1), e30415 (2012).</li></ol>

Çıkar çatışması ilan etti.

Biz cerrahi pankreas örnek insan adacık lazer mikrodiseksiyon (LMD) için güvenilir bir yaklaşım açıklanmaktadır. Bir LMD mikroskop mevcuttur kaydıyla, bu işlem böylece hem non-diyabetik ve tip 2 diyabetik bireylerin insan adacık materyale erişimin artırılması, kısmi pancreatectomies gerçekleştirirken herhangi bir araştırma kurumunda uygulanması olabilir. Bu adacık izolasyonu için sunulan pancreata yetersizliğinden ötürü verilen özellikle önemlidir. Uygun olup açıdan kollajenaz sindirimi ...

<table border="1"><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> 2-metilbutane (izopentan) </td><td> ROTH </td><td> 3927.1 </td><td></td></tr><tr><td> AdhesiveCap (opak, 500 ul) </td><td> ZEISS </td><td> 415190-9201-000 </td><td></td></tr><tr><td> Cellstar Borular (50 ml) </td><td> Greiner Bio-one </td><td> 210 261 </td><td> desteği etek ile </td></tr><tr><td> Cellstar Borular (50 ml) </td><td> Greiner Bio-one </td><td> 227.261 </td><td></td></tr><tr><td> Dietil pyrocarbonate (DEPC) </td><td> SIGMA </td><td> D 5758 </td><td></td></tr><tr><td> Donmuş karbondioksit </td><td></td><td></td><td></td></tr><tr><td> Mutlak etanol </td><td> VWR </td><td> 20821.310 </td><td></td></tr><tr><td> Sıvı azot </td><td></td><td></td><td></td></tr><tr><td> Boya fırçasını </td><td></td><td></td><td></td></tr><tr><td> PALM MicroBeam </td><td> ZEISS </td><td></td><td></td></tr><tr><td> Peel-a-yol gömme kalıpları (kesilmiş), 12 x 12 mm </td><td> ProSciTech </td><td> RR12 </td><td> En içten 22x22 mm, derinlik 21 mm </td></tr><tr><td> Arcturus PicoPure Dondurulmuş RNA İzolasyonu Takımı </td><td> Applied Biosystems </td><td> KIT 0204 </td><td></td></tr><tr><td> Plastik kelepçe </td><td></td><td></td><td></td></tr><tr><td> Ustura </td><td></td><td></td><td></td></tr><tr><td> RNaz-Alerjik DNaz Seti </td><td> Qiagen </td><td> 79254 </td><td></td></tr><tr><td> RNaseZAP </td><td> SIGMA </td><td> R 2020 </td><td></td></tr><tr><td> Bisturi /cerrahi bıçak </td><td> Techno Cut </td><td> 2800111 </td><td></td></tr><tr><td> SuperFrost Plus yapışma mikroskop slaytlar </td><td> Thermo Scientific </td><td> J1800AMNZ </td><td> 25x75x1.0 mm </td></tr><tr><td> Tissue-Tek Ekim Bileşik </td><td> Sakura </td><td> 4583 ya da 0807000022 </td><td></td></tr><tr><td> Cımbız </td><td> BRAUN </td><td> BD168R </td><td></td></tr><tr><td> Ksilen </td><td> VWR </td><td> 28975.291 </td><td></td></tr></table>

improved protocol for laser microdissection of human pancreatic islets from surgical specimens

Lazer mikrodisseksiyon (LMD) parankim 1,2 dakika miktarlarda seçili hücrelerin ve dokuların iyileşme sağlayan bir tekniktir. Disseke hücreleri gibi Transkriptomik veya proteomik çalışmalar, DNA değerlendirmesi veya kromozom analizi 2,3 gibi soruşturmaların, çeşitli için kullanılabilir. LMD ait özellikle zor bir uygulama RNA 4 değişkenlik nedeniyle, hücreler örneğin pankreas gibi RNaz, yönünden zengin olan dokulardan gelen parçalanmış zaman özellikle belirgin olabilir, transcriptome analizdir. Hedef hücrelerin hızlı belirlenmesi ve toplanmasını sağlayan bir mikrodisseksiyon protokol doku işleme süresini kısaltmak için ve, sonuç olarak, RNA koruma sağlamak amacıyla, bu durumda önemlidir. Burada Transkriptomik çalışmalar 5 için kullanılmak üzere cerrahi örneklerden insan pankreatik beta hücreleri elde etmek için bir protokol açıklar. 0.5-1 hakkında c pankreas Küçük parçalarm 3 kesit kadar hemen 2-metilbutane soğutulmuş dondurulmuş Tissue-Tek Ekim Bileşik gömülü rezeke pankreas numunelerin sağlıklı görünen marjları, kesilmiş ve -80 ° C&#39;de depolanan edildi. 10 mikron kalınlığında, kırk seri bölümleri 1-2 dk kriyostat içinde kurutulur ve -80 saklanan cam slaytlar, ° C için ayrı ayrı transfer, -20 ° C ayar altında kriyostat üzerine kesildi Hemen lazer mikrodiseksiyon işlem öncesi, bölümleri buz% 100 iki bir dakikalık inkübasyonlar tarafından, buz giderilen taze 30 sn için% 70 etanol hazırlanan buz DEPC-işlenmiş su 5-6 dips ile yıkandı ve susuz edildi Etanol 4 dk için ksilen (doku kurutma için kullanılan) takip etmektedir; doku bölümleri 3-5 dakika süreyle daha sonra o zaman hava kurutuldu. Önemli olarak, bütün adımları, ksilen içinde kuluçka dışında, buz gibi soğuk reaktifler kullanılarak yapıldı - daha önce açıklanan protokol 6 fazla bir değişiklik. Utbuz reaktiflerin yonu beta hücrelerinin içsel otofloresans belirgin bir artışa yol açmıştır, ve bunların karşılaşılmaktadır. Mikrodisseksiyon için, dört bölümden her zaman susuz: iki nem ve ağartma gelen doku korumak için, bir folyo sarılı 50 ml tüp içine yerleştirildi, geriye kalan iki hemen microdissected edildi. Bu prosedür (AutoLPC) modu catapulting Otomatik Lazer Basınç kullanan bir PALM MicroBeam enstrüman (Zeiss) kullanılarak yapıldı. 40-60 dk daha artık gerekli dört cryosections gelen beta hücre / adacık diseksiyon tamamlanması. Hücreler bir AdhesiveCap içine toplanmış ve 10 ul lizis tamponu ile lizlendi. Transkriptomik analiz için her biri tek bir RNA örnek 10 hücre microdissected örnekleri, Pico Saf RNA izolasyon kiti (Arcturus) kullanarak, RNA ekstre bir araya getirilmesiyle elde edilmiştir. Bu protokol böylece hızlı ve doğru bir şekilde tanınmasını ve c kolaylaştırılması, insan beta hücrelerinin içsel otofloresans artırırollection. Bu prosedürü daha da iyileştirilmesi tip 2 diyabet ile ilişkili değişiklikler daha iyi anlamasını mümkün etkileri olan, fenotipik farklı beta hücrelerinin diseksiyonu sağlayabilir.

Şekil 1 &#39;de gösterildiği gibi, modifiye dehidrasyon protokol daha önce yayınlanmış protokol 6&#39;ya göre beta hücrelerinin otofloresans bir iyileşmeye yol açmıştır. Açıklanan protokol uygulama, 39 cerrahi pankreas örneklerin her biri 31&#39;544&#39;704 mikron 3 doku / pankreas numunenin bir ortalaması (- 81&#39;522&#39;153 um 3 8&#39;742&#39;390 aralığı) için seri cryosections 40 oluşturmak için kullanılan Tablo 1 de göster...

Watch this Scientific Journal Video about Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens at JoVE.com

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Lazer mikrodisseksiyon parankiminde dakika miktarlarda seçili hücrelerin iyileşme sağlayan bir tekniktir. Burada Transkriptomik çalışmaları için kullanılacak cerrahi yöntemlerle alınan insan pankreas adacık elde etmek için bir protokol açıklar. Tüm protokol böylece toplama kolaylaştırılması insan beta hücrelerinin içsel otofloresansı, geliştirir.

Cerrahi Örnekler kaynaktan İnsan pankreas adacıklarının Lazer Diseksiyon için Geliştirilmiş Protokolü

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

improved-protocol-for-laser-microdissection-human-pancreatic-islets

Research

JoVE Journal

Medicine

14.3K Görüntüleme.  Paul Langerhans Institute Dresden. Lazer mikrodisseksiyon parankiminde dakika miktarlarda seçili hücrelerin iyileşme sağlayan bir tekniktir. Burada Transkriptomik çalışmaları için kullanılacak cerrahi yöntemlerle alınan insan pankreas adacık elde etmek için bir protokol açıklar. Tüm protokol böylece toplama kolaylaştırılması insan beta hücrelerinin içsel otofloresansı, geliştirir.

Video: Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

Cerrahi Örneklerinin tümör eksplantlarındaki Roman Anti-Kanser İlaç Geliştirme Yöntemi


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

Tıp

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Kısmen Pancreatectomized hastalar İnsan Islets izolasyonu

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

İnsan Pankreas için Toplama Protokolü

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

İnsan Pankreas Adacıklar için boyama Protokoller

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Hepatik Arter İmar Kısmi Ortotopik Karaciğer Nakli Bir Rat Modeli Cerrahi İşlemleri

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Retinal detachment (RD) describes a separation of the neurosensory retina from the retinal pigmented epithelium (RPE). The RPE is essential for normal function of the light sensitive neurons, the photoreceptors. Detachment of the retina from the RPE creates a physical gap that is filled with extracellular fluid. RD initiates cellular and molecular adverse events that affect both the neurosensory retina and the RPE since the physiological exchange of ions and metabolites is severely perturbed. The consequence for vision is related to the duration of the detachment since a rapid reapposition of the two tissues results in the restoration of vision 1. The treatment of RD is exclusively surgical. Removal of vitreous gel (vitrectomy) is followed by the removal non essential part of the retina around the detached area to favor retinal detachment. The removed retinal specimens are res nullius (nothing) and consequently normally discarded. To recover RNA from these surgical specimens, we developed the procedure jouRNAl that allows RNA conservation during the transfer from the surgical block to the laboratory. We also standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for global gene expression analysis. The quality of the RNA was validated both by RT-PCR and microarray analysis. Analysis of the data shows a simultaneous involvement of inflammation and photoreceptor degeneration during RD.

We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis.

Journal kullanarak İnsan Retina Cerrahi Örneklerinin transkriptomik Analizi

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

Klinik Olarak İlgili Hayvan Modellerinde ve 3D İmmün entrikalarda Kullanılmak Üzere Cerrahi Örneklerden Yüksek Dereceli Glioma Hücre Kültürünün Optimizasyonu

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Histerektomi Örneklerden İlköğretim İnsan endometrial stroma hücrelerinde Kurulması İçin İki Yöntem

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

RNA Analizi İnsan Prostat Epitelinin Lazer yakalama mikrodiseksiyon

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon dioxide and waste products. The impairment of microvascular vasomotion might be a crucial step in the development of microcirculation-related diseases. In addition, the highly vascularized pancreatic islet is adapted to support endocrine function. In this respect, it seems possible to infer that the functional status of pancreatic islet microvascular vasomotion might affect pancreatic islet function. Analyzing the pathological changes of the functional status of pancreatic islet microvascular vasomotion may be a feasible strategy to determine contributions that pancreatic islet microcirculation makes to related diseases, such as diabetes mellitus, pancreatitis, etc. Therefore, this protocol describes using a laser Doppler blood flow monitor to determine the functional status of pancreatic islet microvascular vasomotion, and to establish parameters (including average blood perfusion, amplitude, frequency, and relative velocity of pancreatic islet microvascular vasomotion) for evaluation of the microcirculatory functional status. In a streptozotocin-induced diabetic mouse model, we observed an impaired functional status of pancreatic islet microvascular vasomotion. In conclusion, this approach for assessing pancreatic islet microvascular vasomotion in vivo may reveal mechanisms relating to pancreatic islet diseases.

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Pancreatic islet microvascular vasomotion regulates islet blood distribution and maintains the physiological function of islet &#946; cells. This protocol describes using a laser Doppler monitor to determine the functional status of pancreatic islet microvascular vasomotion in vivo and to assess the contributions of pancreatic islet microcirculation to pancreatic-related diseases.

Lazer Doppler: pankreas adacık mikrovasküler Vasomotion Vivo içinde ölçüm için bir araç

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...