University of Illinois at Chicago gibi Adacık Hücre Kaynak Merkezi NIH hibe (RFA-RR 05-003), Christopher Vakfı, Efroymson Vakfı ve Tellabs Vakfı fon tarafından desteklenir.

1. Tesis kurulum <ol><li> İnsan adacık izolasyonu, takım çalışması gerektirir bir zaman hassas bir işlemdir ve bu nedenle, dört kişi bu işlemi yapmak için ihtiyaç vardır. Takım masaya ve pankreas cannulates cerrah tarafından yönetilmektedir. </li><li> Adacık izolasyon ekibi UIC adacık izolasyonu tesisi girer dakika başlar. Ekip üyeleri, santrifüj gibi, gerekli tüm enstrümantasyon, kontrol edilir ve açık olduğunu emin olun. Ayrıca, tüm davlumbaz ve yüzeyleri sterilize etmek için emin olun. </li><li> İki kişi kanülasyon tablo, sulu kar makinesi, sindirim devre, perfüzyon ünitesi, COBE arıtma makine ve cerrahi masaları kuracak. </li><li> Aynı zamanda, diğer iki ekip üyeleri, izolasyon sırasında gerekli ortam hazırlayacaktır. </li><li> Sonra, bakteriyoloji tüpleri (şişe, tüp, form ve çanta), numune alma tüpleri (ön yıkama ile dolu) ve değerlendirme tüpleri ve plakaları hazırlanır. </li><li> Aşağıdaki gibi ayarlayın arıtma tüpü hazırlayın: 1. tüp boş ve 150 ml işaretli, tüpler 2-12, 200 ml insan albumini (yıkama solüsyonu) ile desteklenmiş M199 medya ile doldurulmuş ve 230 ml &#39;işaretli, ve nihayet 50 ml konik tüp 50 ml yıkama solüsyonu ile doldurulur. Bu hazırlık adımları biz hazırlanması ve pankreas perfüze başlamaya hazır tam. </li></ol> 2. Pankreas hazırlanması ve perfüzyon <ol><li> Hazırlanması ve pankreasın perfüzyon başlamak için, bir operatör, steril tekniği kullanarak ambalaj organ kaldırır. Cerrah dekontaminasyon tablo pankreas transfer olacak. </li><li> Cerrah orijinal pankreas koruma çözümü ve hazırlanan &quot;İhale&quot; bakteriyoloji şişeleri doldurmak için kullanan bir operatörü, el ile bir adet 60 ml şırınga dolduracaktır. </li><li> Organ adacık izolasyonu (pankreas, satın alma sırasında hasar veya anatomik olarak anormal ise, adacık izolasyonu için kullanılan) için uygun olup olmadığını belirlemek için bir görsel denetim sonra, cerrah kollajenaz hazırlamak için bir operatör soracaktır. Yeniden askıya alınması zaman farklı enzim marka ve birçok arasında değişmektedir unutmayın. </li><li> Cerrah hazır olduğunda, dekontaminasyon tablo (Betadine, Fungizone / Sefazolin ve HBSS) dekontaminasyon çözümler getirmek ve onlara uygun kaplar içine dökün. Cerrah, bir dakika için her dekontaminasyon çözüm pankreas pankreas yıkama dekontaminasyon gerçekleştirir. Pankreas sonra tartılır olacağı küçük steril bir kavanoza konur. Ağırlığı toplu kayıt içine girildikten sonra, pankreas, bazı koruma çözümü ile kanülasyon tavaya konur. </li><li> Cerrah ortaya çıkarmak ve kısmen ana kanalı insizyon ve bir kanül ile pankreas her bir yarısı cannulate. Kanül dikiş tarafından temin edilecektir. Pankreas serpmek için, ilk perfüzyon ünitesi taşımak ve enzim çözeltisi havza içine dökün. Toplam perfüzyon süresi 5 dakika için 180 mmHg, sonra 5 dakika süreyle 80 mmHg, yaklaşık 10 dakika. Pankreas perfüzyon sonra şişmiş. </li><li> Kanülasyon tavaya yağ dokusu, pankreas dikkatli bir şekilde kesilmiş perfüze pankreas, taşıyın. Sonra 10-12 parçalar halinde kesilmiş ve pankreas sindirim için Ricordi Odası transfer. </li></ol> 3. Pankreas sindirim <ol><li> Sindirim adım başlamak için, boş Ricordi Odası pankreas aktarmak, ekranda koymak Pulmozyme 2.5 ml şişe, (odası doku blok büyük bir yığın önlemek) eklemek, oda kapatın ve sistemi doldurun. </li><li> 5 dakika boyunca çözüm pompalama başlayın. Odanın sıcak bir çözüm boyunca eşit dağıtmak için hafifçe sallayın. Beş dakika sonra, tüm sindirim aşaması için odasına yavaşça sallayın. 37 ° ˚ C sıcaklık koruyun </li><li> Rock odasının sıcaklığı 37 °, sonra odasına sallamak ulaşıncaya kadar yavaşça. Bir numune alın ve her iki dakikada bir ücretsiz adacıklar kapsamında olup olmadığını algılayabilir. Toplu kayıt sindirim tabloda adacık değerlendirme bilgileri kaydedin. Adacık yüzdesi% 50 çözüm ücretsiz ve izole edilmiş yüzen kadar izlemeye devam edin. </li></ol> 4. Doku toplama <ol><li> Adacıklar% 50 örneklerinde bulunur, sindirim devresinde soğuk RPMI seyreltme çözüm ekleyerek durdurulur. Doku, 500 ml konik tüpler 30 ml İnsan Albumin ile önceden doldurulmuş toplanan. Tüpler emme süpernatantı kapalı döndü ve pelet soğuk yıkama solüsyonu ile yıkayın. 1 dakika, 4 ˚ C 1100 RPM (dakikadaki dönüş) konik tüpler Spin </li><li> Tek bir 500 ml konik içine sindirilmiş pankreas dokusu ile askıya alma tüpleri birleştirin. Bir tüp, yıkanmış doku dolu olduğunda, spin ve tüm doku birden çok kez yıkanmış kadar yıkama işlemi tekrarlayın. Bu tüp spin ve tek bir 250 ml konik tüp içine doku transferi. 250 ml ile ses getirM199 yıkama solüsyonu HA ile desteklenmiş ve örnekleme için hazırlamak. </li><li> Bir pipet yardımıyla 1 ml örnek alın ve önceden 9 ml yıkama solüsyonu ile dolu bir 15 ml konik tüp ekleyin. Daha sonra bir ızgara sayma çanak içine doğrudan 1ml dışarı yavaşça tersine 15 ml konik tüp ve bu örnek karıştırın. Hücre sayımı ve seyreltme faktörü 2500 (10x250) kullanarak hücre sayısını hesaplamak. </li><li> 250 ml konik tüp Spin ve UW (Wisconsin Üniversitesi) genellikle organ korunması için kullanmak Çözüm 150 ml. UW eklendiğinde, pipet kullanarak hücre süspansiyonu iyice karıştırın. UW başlangıç ​​zamanını kaydedin ve arıtma operatörü iletişim. Buz ve bazen girdap tüp (ler) bırakın. Şimdi adacıklar arındırılmalıdır. </li></ol>

<ol>
	<li>Shapiro, A. 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=Islet+transplantation+in+seven+patients+with+type+1+diabetes+mellitus+using+a+glucocorticoid-free+immunosuppressive+regimen.">Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.</a> N Engl J Med. 343, 230-238 (2000).</li><li>Nano, 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=Islet+isolation+for+allotransplantation:+variables+associated+with+successful+islet+yield+and+graft+function.">Islet isolation for allotransplantation: variables associated with successful islet yield and graft function.</a> Diabetologia. 48, 906-912 (2005).</li><li>Barbaro, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+human+pancreatic+islet+purification+with+the+refined+UIC-UB+density+gradient.">Improved human pancreatic islet purification with the refined UIC-UB density gradient.</a> Transplantation. 84, 1200-1203 (2007).</li><li>Lakey, J. R., Rajotte, R. V., Warnock, G. L., Kneteman, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pancreas+preservation+prior+to+islet+isolation.+Cold+ischemic+tolerance.">Human pancreas preservation prior to islet isolation. Cold ischemic tolerance.</a> Transplantation. 59, 689-694 (1995).</li><li>Fridell, J. 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=Comparison+of+histidine-tryptophan-ketoglutarate+solution+and+University+of+Wisconsin+solution+for+organ+preservation+in+clinical+pancreas+transplantation.">Comparison of histidine-tryptophan-ketoglutarate solution and University of Wisconsin solution for organ preservation in clinical pancreas transplantation.</a> Transplantation. 77, 1304-1306 (2004).</li><li>Potdar, S., 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=Initial+experience+using+histidine-tryptophan-ketoglutarate+solution+in+clinical+pancreas+transplantation.">Initial experience using histidine-tryptophan-ketoglutarate solution in clinical pancreas transplantation.</a> Clin Transplant. 18, 661-665 (2004).</li><li>Salehi, P., 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=Human+islet+isolation+outcomes+from+pancreata+preserved+with+Histidine-Tryptophan+Ketoglutarate+versus+University+of+Wisconsin+solution.">Human islet isolation outcomes from pancreata preserved with Histidine-Tryptophan Ketoglutarate versus University of Wisconsin solution.</a> Transplantation. 82, 983-985 (2006).</li><li>Ricordi, C., Lacy, P. E., Scharp, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+islet+isolation+from+human+pancreas.">Automated islet isolation from human pancreas.</a> Diabetes. 38, 140-142 (1989).</li><li>Hanley, S. C., Paraskevas, S., Rosenberg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+and+isolation+variables+predicting+human+islet+isolation+success.">Donor and isolation variables predicting human islet isolation success.</a> Transplantation. 85, 950-955 (2008).</li><li>Toso, 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=Factors+affecting+human+islet+of+Langerhans+isolation+yields.">Factors affecting human islet of Langerhans isolation yields.</a> Transplant Proc. 34, 826-827 (2002).</li></ol>

Izolasyon sonucu 9,10 etkileyen çeşitli faktörler vardır. Bunların arasında, pankreas dokusu sindirim önemli bir rol oynar. Kendi insan adacık izolasyon deneyimine dayanarak, potansiyel adacık verim ve kaliteyi etkileyebilecek kritik noktaları, aşağıda özetlenmiştir. <ol><li> Enzimlerin farklı türleri kullanılmaktadır sindirim süresi farklı. </li><li> En kısa sürede% 40-50 olarak asiner doku tamamen ayrılmış adacıklar, sindirim seyreltme çözüm ekleyerek ve doku soğuma derhal durdurulma...

<table><tbody><tr><td>Ice Slush Machine</td><td>equipment</td><td>Taylor Company</td><td>K4036546</td><td>make sterile ice</td></tr><tr><td>Centrifuge</td><td>equipment</td><td>Beckman Coulter Inc.</td><td>424803</td><td></td></tr><tr><td>MZ6 Inverted Microscopy TLBD4.1</td><td>equipment</td><td>Leica Microsystems</td><td>4103</td><td></td></tr><tr><td>Cell Culture Dish with 2 mm Grid</td><td>equipment</td><td>Nalge Nunc international</td><td>174926</td><td>islet counting</td></tr><tr><td>Digital Water Bath EX7</td><td>equipment</td><td>NesLab</td><td>105361058</td><td></td></tr><tr><td>Thermometer</td><td>equipment</td><td>IBI:IR</td><td>60444</td><td></td></tr><tr><td>MonoBloc Balance AB 104-S</td><td>equipment</td><td>Mettler Toledo</td><td>1119430864</td><td>measure pancreas weight</td></tr><tr><td>MasterFlex II Speed Controller</td><td>equipment</td><td>Cole-Parmer</td><td>J00006445</td><td>adjust flow speed during degestion</td></tr><tr><td>Digital Sight DS L1</td><td>equipment</td><td>Nikon Instruments</td><td>217267</td><td></td></tr><tr><td>Perfusion Unit</td><td>equipment</td><td>Custom Made</td><td>n/a</td><td></td></tr><tr><td>Cooler for perfusion unit</td><td>equipment</td><td>Fisher Scientific</td><td>3013P</td><td></td></tr><tr><td>Ricordi Chamber</td><td>equipment</td><td>University of Miami</td><td>n/a</td><td></td></tr><tr><td>RPMI dilution solution</td><td>reagent</td><td>Mediatech, Inc.</td><td>99783024</td><td></td></tr><tr><td>University of Wisconcin (UW) solution</td><td>reagent</td><td>DURAMED</td><td>1000-46-06</td><td></td></tr><tr><td>Hank&amp;rsquo;s Buffered Salt Solution (HBSS)</td><td>reagent</td><td>Mediatech, Inc.</td><td>99-597-CM</td><td></td></tr><tr><td>25% Human Albumin</td><td>reagent</td><td>GRIFOLS</td><td>NDC 61953-0002-2</td><td></td></tr><tr><td>M199 media (wash solution)</td><td>reagent</td><td>Mediatech, Inc.</td><td>99-784-CM</td><td></td></tr><tr><td>Collagenase NB1 GMP grade</td><td>reagent</td><td>SERVA Electrophoresis</td><td>N0002937</td><td></td></tr><tr><td>Neutral Protease NB1 GMP grade</td><td>reagent</td><td>SERVA Electrophoresis</td><td>N0002936</td><td></td></tr><tr><td>Betadine</td><td>reagent</td><td>Cardinal Health</td><td>29906-004</td><td></td></tr><tr><td>Cefazolin</td><td>reagent</td><td>West-Ward Pharmaceutical</td><td>NDC 0143-9924-90</td><td></td></tr><tr><td>Dithizone</td><td>reagent</td><td>Sigma-Aldrich</td><td>D5130</td><td></td></tr></tbody></table>

human pancreatic islet isolation: part i: digestion and collection of pancreatic tissue

Tip 1 diyabet Yönetimi, yıl içinde 100 milyar dolar maliyet, hem birey ve toplum, külfetli. Glikoz izleme ve yeni insülin formülasyonları yaygın kullanımına rağmen birçok kişi hala yıkıcı ikincil komplikasyonlar gelişir. Pankreas adacık nakli, diyabetik hastalarda normal glukoz kontrolü yakın geri yükleyebilirsiniz<sup&gt; 1</sup&gt;, Yoğun insülin tedavisi ile ilişkili ciddi hipoglisemik atakların riski olmadan. Başarılı adacık nakli için yeterli adacık kitle sağlanması önemlidir. Ancak, donör özelliği, organ alımı ve korunması, izolasyon sonucunu etkileyebilecek<sup&gt; 2</sup&gt;. University of Illinois at Chicago (UIC) geliştirilmiş bir arıtma degrade başarılı bir izolasyon protokolü geliştirildi<sup&gt; 3</sup&gt;. Bu program, 2004 yılı Ocak ayında başlayan ve 300&#39;den fazla izolasyonların Kasım 2008&#39;de yapıldı. Pancreata (UW, Wisconsin veya HTK Üniversitesi, Histidin-Triptofan Ketoglutarate), soğuk muhafaza çözümleri gönderildi<sup&gt; 4-7</sup&gt; Hücre İzolasyonu Laboratuvar adacık izolasyonu için UIC. Pankreas adacık Ricordi tarafından tanımlanan yöntem değiştirilmiş bir versiyonu olan UIC yöntemi ile izole edildi<em&gt; Ve ark.</em&gt;<sup&gt; 8</sup&gt;. Kısaca, çevre doku pankreas temizlendikten sonra, enzim solüsyonu (Serva kollajenaz + Nötr Proteaz veya Sigma V enzim) ile perfüze edildi. Şişmiş pankreas Ricordi sindirim odasına transfer değiştirilmiş bir kapalı dolaşım tüp sistemine bağlı ve 37 ° C&#39;ye kadar ısındı. Sindirim sırasında, oda hafifçe sarsıldı. Örnekleri sindirim ilerlemeyi izlemek için sürekli olarak alınmıştır. Mikroskop altında ücretsiz adacıklar saptandı sonra, sindirim soğuk kızarma tarafından durduruldu (4 ° C) enzim sulandırmak için dolaşım sistemi içine RPMI seyreltme çözeltisi (Mediatech, Herndon, VA). Doku, insan albumini ile desteklenmiş M199 medya toplanır ve yıkandıktan sonra, ön arıtma sayımı için örnek ve arıtma önce UW solüsyonu ile inkübe edildi. İnsan Islets saflaştırılması ve Kültür: Arınma süreci Bölüm II açıklanacaktır.

Watch this Scientific Journal Video about Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue at JoVE.com

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

, Yüksek kalite ve insan adacıklar uygun miktarda sağlamak başarılı bir adacık nakli için önemli önkoşullar biridir. Bu video, adım adım tarif insan pankreas adacık izolasyonu için prosedürler: değiştirilmiş bir otomatik yöntemi kullanılarak (bölüm I sindirim ve pankreas dokusu toplanması).

İnsan Pankreas Adacık İzolasyonu: Bölüm I: Sindirim ve Pankreas Doku Koleksiyon

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

human-pancreatic-islet-isolation-part-i-digestion-collection

Research

JoVE Journal

Biology

19.3K Görüntüleme.  University of Illinois, Chicago. , Yüksek kalite ve insan adacıklar uygun miktarda sağlamak başarılı bir adacık nakli için önemli önkoşullar biridir. Bu video, adım adım tarif insan pankreas adacık izolasyonu için prosedürler: değiştirilmiş bir otomatik yöntemi kullanılarak (bölüm I sindirim ve pankreas dokusu toplanması).

Video: Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

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 ...

Tıp

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Yenidoğan Fare Pankreas gelen Fonksiyonel Adacık etkili İzolasyonu

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Gelişim Biyolojisi

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

Insan pankreatik Izletinin tek hücreli RNA sıralaması ve Analizi

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Genetik

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

İnsan Pankreas Adacık İzolasyonu: Bölüm II: İnsan Islets saflaştırılması ve Kültür

Biyoloji

Pancreatic Tissue Dissection to Isolate Viable Single Cells

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for approximately 90% of the pancreas and includes cells that produce and secrete digestive enzymes. The digestive enzymes are produced in the pancreatic acinar cells, stored in vesicles called zymogens, and are then released into the duodenum via the pancreatic duct to initiate metabolic processes. The enzymes produced by the acinar cells can kill cells or degrade cell-free RNA. In addition, acinar cells are fragile, and common dissociation protocols result in a large number of dead cells and cell-free proteases and RNases. Therefore, one of the biggest challenges in pancreatic tissue digestion is recovering intact and viable cells, especially acinar cells. The protocol presented in this article shows a two-step method that we developed to meet this need. The protocol can be used to digest normal pancreata, pancreata that include pre-malignant lesions, or pancreatic tumors that include a large number of stromal and immune cells.

Pancreatic metaplastic cells are precursors of malignant cells that give rise to pancreatic tumors. However, isolating intact viable pancreatic cells is challenging. Here, we present an efficient method for pancreatic tissue dissociation. The cells can then be used for single-cell RNA sequencing (scRNA-seq) or for two- or three-dimensional co-culturing.

Canlı Tek Hücreleri İzole Etmek için Pankreas Doku Diseksiyonu

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for ...

Kanser Araştırmaları

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced &#946; cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the &#946; cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

Bir fare pankreas adacık hücre tabanlı tarama diyabetojenik çevresel kimyasallar için

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

İmmünoloji ve Enfeksiyon

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

Bir Yüksek içerik<em&gt; İn Vitro</em&gt; Pankreas Adacık β-hücre çoğaltma Keşif Platformu

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Farelerden Pankreas Adacık İzolasyon için Basit Bir Yüksek Verimlilik Protokolü

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

<meta charset="utf8"></head><body>Domuz pankreasının cerrahi perfüzyonu ile domuz adacıklarının saflaştırılması

Surgical Perfusion and Isolation of the Porcine Pancreas for Islet Isolation

Pancreatic islet transplantation is an emerging treatment for type I diabetes; however, it is limited by donor matching and availability. Porcine islet xenotransplantation offers a promising alternative to allotransplantation, with the potential for large-scale production of on-demand, functional islets. The yield and viability of isolated islets is highly susceptible to the quality of the donor pancreas and the method of procurement, particularly the duration of warm-ischemia time. To improve organ preservation and subsequent islet yield and viability, we have developed a protocol for surgical perfusion and resection of the porcine pancreas. This protocol employs direct infrarenal aortic cannulation and organ perfusion to both minimize warm-ischemia time and simplify the procedure for operators who do not have extensive surgical expertise. Subsequent arterial perfusion of the pancreas via the aorta flushes stagnant blood from the microvasculature, thereby reducing thrombosis and oxidative damage to the tissue. This manuscript provides a detailed protocol for surgical perfusion and resection of the porcine pancreas, followed by islet isolation and purification.

<meta charset="utf8"></head><body>Yazar Spotlight: Ksenotransplantasyon için Adacık Saflaştırmada Teknikler ve Zorluklar

This protocol provides a step-by-step guide for the procurement of a porcine pancreas for islet isolation and purification.

Adacık izolasyonu için domuz pankreasının cerrahi perfüzyonu ve izolasyonu

Pancreatic islet transplantation is an emerging treatment for type I diabetes; however, it is limited by donor matching and availability. Porcine islet ...

İnsan Pankreas Adacık İzolasyonu: Bölüm I: Sindirim ve Pankreas Doku Koleksiyon

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İnsan Pankreas Adacık İzolasyonu: Bölüm II: İnsan Islets saflaştırılması ve Kültür

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Bir fare pankreas adacık hücre tabanlı tarama diyabetojenik çevresel kimyasallar için

Bir Yüksek içerik

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