Bu çalışma Det sutable Forskningsråd / Sundhed og Sygdom Bir Genetik Pilot ödülü NCCR Frontiers, Juvenil Diyabet Araştırma Vakfı Hibe 41-2009-775 ve Grant 12-126875 tarafından sırayla finanse edildi. Yazarlar video çekimi sahipliği için Spagnoli laboratuvar teşekkür ederim.

Bu protokol kemirgen E10.5 türetilen pankreas organoids epitel hücreleri pankreas ayrışmış büyümeyi hedeflemektedir. Protokol hayvan deney için etik kurul onayı gerektirir. E10.5 fare embriyoları Dorsal Pankreas Bud 1. Diseksiyon <ol><li> Bir makas ile karın açmak, embriyonik gün (E) 10.5 aşımına hamile fareler kurban, iki rahim boynuzları çıkarın ve soğuk fosfat tamponu (PBS) veya Dulbeccol ile dolu bir 10 cm Petri kabındaki koyun modifiye temel, orta (DMEM) buz üzerinde tutuldu. Hücre ekimi için kurban gelen toplam deney hücre zarar görmesini önlemek için, 60-90 dakika içinde yapılır. </li><li> Küçük bir makas kullanarak tek tek embriyo bölümler halinde rahim ayırın. Gibi soğuk PBS ile bir 35 mm Petri kabı, bir embriyo transfer ve üstten aydınlatma ile bir mikroskop altında görselleştirmek. Diseksiyon forseps ile çevreleyen kas, desidua ve yumurta sarısı sa kaldırmakc ve embriyo maruz. Lütfen soğuk DMEM ortama embriyo yerleştirin. Embriyolar kolayca 3 ml&#39;lik plastik transferi pipet / damlalık kullanarak transfer edilebilir. Devam etmeden önce benzer bir şekilde, her bir embriyonun izole edin. </li><li> PBS içinde temiz bir 35 mm Petri kabı içine bir embriyo yerleştirin. <ol><li> Ön ayakları kaldırmak için ince forseps (0.05 mm genişlik) kullanın. Yavaşça omurilik bölgesinden sindirim sistemi (Şekil 1) ayırmak için açılış forseps yerleştirin. İSTEĞE BAĞLI: daha rahat, mide görmek aşağı sarısı sap altında kalp ve kuyruk bölgeye, embriyonun üst vücut kaldırmak için. </li><li> Mide, karaciğer ve bağırsak bulun. Forseps kullanılarak, bağırsak mideden mide-bağırsak yolu izole etmek ve (Şekil 1), buz üzerinde, soğuk DMEM içine yerleştirin. Dorsal pankreatik bud mide dorsal, posterior (Şekil 1) takılır. </li><li> Bir simi her bireyin gastrointestinal izoleDevam etmeden önce lar şekilde. Embriyoların genotip gerekiyorsa, diseksiyon zamanda kuyruk toplar ve buz soğuk DMEM ile 24 kuyucuklu bir plaka içerisinde farklı bir kuyu içinde her bir mide-bağırsak yolu tutmak. </li></ol></li><li> Soğuk PBS ile temiz bir 35 mm Petri tabağına yerleştirin, bir mide-bağırsak yolu. Şimdi diseksiyon mikroskop altında dorsal pankreas tomurcuk görselleştirmek ve teşrih parlak alanda aşağıdan aydınlatma kullanın. Bu koşullar hacimlerinin görselleştirme optimize eder. Elektrolitik-bilenmiş tungsten iğne veya 20 G şırınga iğneleri kullanarak, epiteli etrafında mümkün olduğunca az mezenşimin ile pankreas tomurcuğu (Şekil 1) izole. <ol><li> 2-3 dakika boyunca, bir soğuk dispase çözeltisi (1.25 mg / ml) ihtiva eden bir petri çanağı içine izole edilmiş tomurcuk aktarın. Bu noktadan itibaren, transfer en uygun bir ağızdan-kontrol edilen esnek bir boru bağlanmış alev çekilmiş 50 ul bir cam kılcal ile yapılabilir. Alternatif olarak, ancak daha ritomurcuğu kaybetme sk, uygun plastik ipuçları ile 10 ul otomatik pipet (Pipetman) kullanın. </li><li> Mikroskopik kontrol altında pankreas tomurcuk aspirasyon ve fırlamasını gerçekleştirin. Geri PBS içinde pankreas tomurcuk koydu. Bundan başka, cam kılcal (Şekil 1) kullanılarak, iğnelerin ve yumuşak aspirasyon ile mezenşimin gelen izole edilmiş pankreas tomurcuk temizleyin. </li><li> Tüm mezenşimin çıkarıldığında, soğuk PBS içinde pankreatik tomurcuk durulama; (60-iyi mini tepsi soğuk DMEM 10 ul ile dolu) bireysel kuyularda soğuk DMEM&#39;ye her tomurcuk transfer. Bu doku çok yapışkan yapar, dispase yılında mezenşimi kaldırmak için önemli değildir. </li></ol></li></ol> Dağılmış hücreler 2. Kaplama ve Kültür <ol><li> Durulama için 10 ul PBS ile dolu 60-iyi mini kasetlerinden konik kuyuların içine alev-kapiller çekti cam ile tüm embriyolar disseke epitelyaya aktarın. <ol><li> Tripsin% 0.05 a 10 ul transfer tomurcuknd bu 4 dakika süreyle 37 ° C&#39;de inkübe edin. 10 ul DMEM +% 10 fetal dana serumu (FCS) ile bir kuyuya tomurcuk aktararak tripsin etkisiz hale getirirler. </li><li> Bir pipet çektirmesi ile çekilmiş ince kılcal damar yoluyla aspirasyon ile hücre süspansiyonu ayrışır. Bu hücreleri ayırmak için yukarı ve aşağı pipetleme sırasında bu aşamada kabarcıkları önlemek için önemlidir. Pankreas organoids optimum 5-15 hücre küçük gruplar başlar ve bu nedenle kısmi ayrılma (Şekil 1) kullanılması tavsiye edilir. </li></ol></li><li> Tek tek işleme nedeniyle farklılıkları en aza indirmek amacıyla bir Eppendorf tüpü içine birkaç embriyolarından hücreleri havuzda toplayın. , 1:3 oranında Matrigel soğutulmuş hücre süspansiyonu seyreltin. Kısım 96 oyuklu bir plaka, 8 ul / oyuk veya görüntüleme için optimize edilmiş bir plaka içinde (aşağıya bakınız) için bu karışımı ile gerçekleştirilmektedir. </li><li> Matrigel kalınlaştırmak için izin veren, 5 dakika boyunca 37 ° C&#39;de inkübe edin. Seçim ortamı 70 ul ile kuyu doldurun (organoid veya küre, sekmesine bakınles 1 ve 2) ve 37 ° C de% 5 CO2 ve% 95 hava içeren nemlendirilmiş bir ortamda bırakmak </li><li> Her 4. günde orta yerine. Günlük büyüyen pankreas koloniler izlemek ve görüntüleme işlemini belgelemek. </li><li> Daha önce rapor edildiği gibi, 28 çıkar küçük moleküller ya da proteinler, girişim deneyleri için bu aşamada ortamına ilave edilebilir. </li></ol> Tablo 1: organoid ortamı. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Malzeme Adı </td><td> Hazır Konsantrasyon </td><td> Son ortam içinde konsantre </td><td> Stokunun hacmi </td></tr><tr><td> Penisilin-Streptomisin </td><td> % 100 </td><td> % 1 </td><td> 50 ul </td></tr><tr><td> Nakavt Serum değiştirme (ek) </td><td> % 100 </td><td> % 10. </td><td> 500 ul </td></tr><tr><td> 2-merkaptoetanol </td><td> 14.3 M </td&gt; <td> 0.1 mM </td><td> 1 ul </td></tr><tr><td> Forbol Miristat Asetat (PMA) </td><td> 16 iM </td><td> 16 nM </td><td> 5 ul </td></tr><tr><td> Y-27632 (ROCK inhibitörü) </td><td> 50 mM </td><td> 10 uM </td><td> 1 ul </td></tr><tr><td> EGF </td><td> 50 ug / ml </td><td> 25 ng / ml </td><td> 2.5 ul </td></tr><tr><td> Rekombinant İnsan 1 R-spondin </td><td> 250 ug / ml </td><td> 500 ng / ml </td><td> 10 ul </td></tr><tr><td> - Ya da - </td><td></td><td></td><td></td></tr><tr><td> Rekombinant Fare R-1 spondin </td><td> 250 ug / ml </td><td> 500 ng / ml </td><td> 10 ul </td></tr><tr><td> Rekombinant İnsan FGF1 (aFGF) </td><td> 100 ug / ml </td><td> 25 ug / ml </td><td> 1.25 ul </td></tr><tr><td> Heparin (Liquemin) </td><td> 2500 U / ml </td><td> 2.5 U / ml </td><td> 2 ul </td></tr><tr><td> Recombinant İnsan FGF10 </td><td> 100 ug / ml </td><td> 100 ng / ml </td><td> 5 ul </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4,412.25 ul </td></tr><tr><td> Toplam </td><td></td><td></td><td> 5000 ul </td></tr></tbody></table> Tablo 2: Küre ortamı. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Malzeme Adı </td><td> Hazır Konsantrasyon </td><td> Son ortam içinde konsantre </td><td> Stokunun hacmi </td></tr><tr><td> Penisilin-Streptomisin </td><td> % 100 </td><td> % 1 </td><td> 50 ul </td></tr><tr><td> B27 x50 (ek) </td><td> % 100 </td><td> % 10. </td><td> 100 ul </td></tr><tr><td> Rekombinant İnsan FGF2 (bFGF) </td><td> 100 ug / ml </td><td> 64 ng / ml </td><td> 3.2 ul </td></tr><tr><td> Y-27632 (ROCK inhibitörü) </td><td> 50 mM </td><td> 10 uM </td><td> 1 ul </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4845,8 ul </td></tr><tr><td> Toplam </td><td></td><td></td><td> 5000 ul </td></tr></tbody></table> Organoid Geliştirme İlerlemesi 3. Görüntüleme <ol><li> Görüntü organoids günlük veya zaman atlamalı mikroskopi bir floresan time-lapse mikroskop kullanılarak. Zaman atlamalı görüntüleme için, bir XY (Z) otomatik ters floresan mikroskop kullanın. </li><li> 4-çukurlu plakalara veya 2-5 ml ortam ile doldurulmuş olan cam-tabanlı plakalar içerisinde 3 ul&#39;lik Deposit küçük damlacıklar halinde. 10x uzun mesafe hedefi ile görüntü. Not: floresan izleyiciler ifade eden transjenik farenin kullanılabilir. Örneğin Movie 1 gösterileri Pdx1-Ngn3-ER TM-ires-nGFP + farelerden 4 organoids ilk genişleme. Nükleer GFP tek nesneleri ancak benzer ilkeler zar floresan ile hücreleri izlemek için uygulanabilir olarak hücreleri izlemek için sağlar bumT / mG fareler 29 gibi. </li><li> Odak sürükleniyor önlemek ve elle tanımlanmış pozisyonlarda 3 veya daha fazla gün için 1 resim / saat almak için yazılım kontrol otomasyon ayarlamak için tohum hücreleri sonra time-lapse görüntüleme 3 saat başlayın. Her bir konumu için, floresan markör ifade raporlama, diferansiyel girişim kontrast (DIC) görüntü hem de GFP sinyali elde. </li></ol> Histoloji için Organoids 4. Recovery <ol><li> Buz üzerinde 96 oyuklu plaka koyun ve buz gibi soğuk PBS ile değiştirilmesi, orta çıkarın. Bu kısmen Matrigel depolimerize. </li><li> Yavaşça genel yapısını bozmayacak amacıyla 1.000 ul ucu kullanarak çevredeki Matrigel kaldırarak, her organoid aspire. Buz gibi soğuk PBS ile bir kuyuya her organoid aktarın. Buz üzerinde plaka tutun. Matrigel Doğrudan tespit de mümkündür. </li><li> ,% 4 paraformaldehit (PFA) içerisinde 15 dakika boyunca organoid saptamak sukroz içinde dondurularak saklanabilmesi ve jelatin içine gömmek. Her Süreçorganoid önce (Johansson et al., 2007) tarif edildiği gibi 4 cryosectioning ve histoloji için. </li></ol> PCR ve Biyokimya Organoids 5. Recovery <ol><li> Buz üzerinde 96 oyuklu plaka yerleştirin ve orta çıkarın. Hücresel RNA stabilize etmek ve korumak için oyuk başına RNAlater 60 ul ekle. </li><li> Kısmen buz üzerinde depolymerizing her iyi mekanik olarak jel bozar. Genel yapısını bozabilir veya 200 ul ucu ile mekanik jel bozarak (Matrigel ile) tüm iyileşmez için 1.000 ul uç kullanarak bireysel organoids kurtarmak ya. Bir RNAse serbest olmayan yapışkan Eppendorf tüpüne da içerik aktarmak için 1.000 ul ucu kullanın buz üzerinde tutulmalıdır. </li><li> 60 ul RNAlater ile kuyu yıkayın ve aynı Eppendorf tüpüne kalan içerik eklemek. </li><li> 4 ° C &#39;de 500-1000 x g&#39;de 5 dakika boyunca tüpleri Spin </li><li> Sadece 20-30 ul bırakarak, süpernatant kaldırmakbirlikte pelet ile tüp içinde RNAlater. Biyokimya için, mümkün olduğu kadar kuru numune saklayın. </li><li> Hemen RNAlater örnekleri dondurma etmeyin; 4 ° CO / N de mağaza (RNAlater iyice dokuya nüfuz sağlamak için). Doku, uzun süreli depolama için -20 ° C &#39;de muhafaza edilebilir ve daha sonra, doku bozulması ve RNA küçük miktarlarda çıkarılması için işlenebilir. </li></ol>

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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=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 10, (2013).</li><li>Greggio, 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=Artificial+three-dimensional+niches+deconstruct+pancreas+development+in+vitro.">Artificial three-dimensional niches deconstruct pancreas development in vitro.</a> Development. 140, 4452-4462 (2013).</li><li>Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45, 593-605 (2007).</li></ol>

The authors have nothing to disclose.

In vitro fonksiyonel beta hücrelerinin büyük ölçekli üretim hala 1 etkisizdir. Bu zorlu bağlamda, gelişim biyolojisi çalışma fonksiyonel beta hücrelerinin farklılaşması için gerekli olan tam sinyalleri deşifre yardımcı olabilir. Bu protokol, in vitro embriyo pankreas progenitörlerin bakım, genişleme ve farklılaşması için izin verir. Bu, diğer endokrin hormon eş bildirilmemektedir insülin üreten beta hücrelerinin oluşumunu içeren, Pdx1 yüksek düzeyde, insül...

Organ kültür vivo araştırmalarda karmaşık ama son derece alakalı arasındaki boşlukları ve hücre çizgi modellerin uygun ama yaklaşık simülasyon köprü kullanışlı bir model sağlar. Pankreas durumunda, endokrin ve ekzokrin hücreleri taklit hücre çizgileri bulunmaktadır transforme edilir, ancak pankreas progenitörlerin mükemmel eşdeğer herhangi bir hücre hattı yoktur. Yetişkin Bütün pankreas kültüre olamaz; izole edilmiş endokrin adacık hücresi çoğalması olmadan birkaç hafta muhafaza edilebilir ve doku dilimleri birkaç saat 5 için in vitro olarak tutulabilir. Embriyonik pankreas kültürü yaygın gelişimini incelemek için sadece kullanılır olmuştur, ama aynı zamanda 8 işler görüntüye, epitelyal-mezenkimal etkileşimleri 4,6,7 araştırmak veya kimyasal onlarla 9 ile müdahale. İki organ kültürü yöntemleri ağırlıklı olarak kullanılır: İlk conve fibronektin kaplı plakalar 2, üzerinde pankreas tomurcukları kültürleme oluşurGörüntüleme amacıyla nient; İkinci seçenek kültürüne en morfogenetiğine korur hava-sıvı arayüzü 3,4 de filtrelerde organ olduğunu. Çok faydalı olmasına rağmen, bu yöntemler, düzleşme belli bir dereceye kadar yol; normal gelişimi ile karşılaştırıldığında ve başlangıç ​​popülasyonu pankreas hücreleri ve mezenkimal hücreler her türlü içeren karmaşık olduğu gibi progenitörlerin genişleme çok sınırlıdır. Kültür yeteneği ve dağınık birincil hücreleri genişletmek soy ilişkileri incelemek ve izole hücre tiplerinin 10 içsel özelliklerini ortaya çıkarmak için değerlidir. Sugiyama ve ark. 11. pankreas atalarıdır ve besleyici katmanlar kültürde 3-5 gündür bazı fonksiyonel karakterleri korunur endokrin atalarıdır korumak olabilir. Atalarıdır doğası / kök hücrelerin rağmen neurospheres 12 ve 13 mammospheres benzer Pancreatospheres, yetişkin adacık ve duktal hücrelerinden genişletilmiştirBu küreler oluşturmak olduğu açık değildir. Buna ek olarak, fizyolojik gelişimi ile aksine, bazı pancreatospheres nöronlar 14,15 ihtiva etmiştir. Küreler yakın zamanda embriyonik pankreas atalarıdır 16,17 ve iyi progenitör genişleme ve sonraki farklılaşma ile pankreata 18 yenileyici üretilir ama morfogenetiğine özetlemek başarısız olduğu görülmüştür. Minyatür organları içine kendi kendini düzenleyebilir ve genellikle dağınık tanımlanan hücrelerden 3 boyutlu modeller son gelişti ve bu bağırsak 19,20, 21, mide, karaciğer 22, prostat 23 ve nefes borusu gibi birden fazla organların gelişimi veya yetişkin devir taklit adres 24. Optik bardak 25, 26 ya da bağırsak, beyin 27 olduğu gibi, bazı durumlarda, gelişme morfolojisi ve farklılaşması, ES hücrelerinden in 3D değinmeyecek edilmiştir. Burada, biz desOnlar farklılaştırmak ve öz-örgütlenmeyi bir 3D Matrigel iskele ayrışmış multipotent pankreas atalarıdır genişletmek için bir yöntem cribe.

<table><tbody><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15070-063</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>KnockOut Serum replacement (supplement)</td><td>Gibco</td><td>10828-028</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>2-mercaptoethanol</td><td>Sigma Aldrich</td><td>3148-25ML</td><td>Stock kept at 4 &amp;deg;C</td></tr><tr><td>Phorbol Myristate Acetate (PMA)</td><td>Calbiotech</td><td>524400-1MG</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>Y-27632 (ROCK inhibitor)</td><td>Sigma Aldrich</td><td>ab120129</td><td>Stock kept at -20 &amp;deg;C. Attention! Stability/source is a frequent source of problems.</td></tr><tr><td>EGF</td><td>Sigma Aldrich</td><td>E9644-2MG</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>Recombinant Human R-spondin 1</td><td>R&amp;amp;D</td><td>4645-RS-025/CF</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>Recombinant Mouse R-spondin 1</td><td>R&amp;amp;D</td><td>3474-RS-050</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>Recombinant Human FGF1 (aFGF)</td><td>R&amp;amp;D</td><td>232-FA-025</td><td>Stock kept at -80 &amp;deg;C - do not include to increase beta cell production</td></tr><tr><td>Heparin (Liquemin)</td><td>Drossapharm</td><td></td><td>Stock kept at 4 &amp;deg;C</td></tr><tr><td>Recombinant Human FGF10</td><td>R&amp;amp;D</td><td>345-FG-025</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>DMEM/F-12</td><td>Gibco</td><td>21331-020</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15070-063</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>B27 x50 (supplement)</td><td>Gibco</td><td>17504-044</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>Recombinant Human FGF2 (bFGF)</td><td>R&amp;amp;D</td><td>233-FB-025</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>Matrigel</td><td>Corning</td><td>356231</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>Trypsin 0.05%</td><td>Gibco</td><td>25300-054</td><td>Stock kept at 4 &amp;deg;C</td></tr><tr><td>RNAlater - RNA stabilizing reagent</td><td>Qiagen</td><td>76104</td><td>Store at RT</td></tr><tr><td>Dispase&amp;nbsp;</td><td>Sigma Aldrich</td><td>D4818-2MG</td><td>Working concentration: 1.25 mg/ml. Stock kept at -20 &amp;deg;C</td></tr><tr><td>BSA for reconstitution</td><td>Milipore</td><td>&lt;a href=&quot;http://www.millipore.com/catalogue/item/81-068-3&quot;&gt;81-068&lt;/a&gt;</td><td>For reconstituition of cytokines&amp;nbsp; - stock kept at -20 &amp;deg;C</td></tr><tr><td>Fetal calf serum (FCS)</td><td>Gibco</td><td>16141079</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>60-well MicroWell trays</td><td>Sigma Aldrich</td><td>M0815-100EA</td><td></td></tr><tr><td>4-well plates</td><td>Thermo Scientific</td><td>176740</td><td></td></tr><tr><td>95-well plates F bottom</td><td>Greiner Bio</td><td>6555180</td><td></td></tr><tr><td>Glas bottom plates</td><td>Ibidi</td><td>81158</td><td></td></tr><tr><td>Disposal glass micropipettes</td><td>Blaubrand</td><td>708745</td><td></td></tr><tr><td>Microscope</td><td></td><td></td><td>Cell&amp;#174; imaging station (motorized inverted Olympus IX81 stand) equipped with a Hamamatsu ORCA ER B7W camera and the Ludin Cube and Box.&lt;br /&gt;
Leica DMI6000 B stand surrounded with a Ludin Cube and Box, equipped with a Leica DFC365 FX camera and the AF6000 Expert/Matrix software command interface.</td></tr><tr><td>Objective</td><td></td><td></td><td>Olympus UPLAN FL NA 0.30 air 9.50 mm 10X long distance;&lt;br /&gt;
Leica HC PL FLUOTAR NA 0.30 air 11.0 mm 10X long distance</td></tr></tbody></table>

in vitro pancreas organogenesis from dispersed mouse embryonic progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

3D Matrigel&#39;de ayrışmış ve seribaşı E10.5 dorsal pankreas atalarıdır pankreas gelişimini özetlemek. Atalarıdır en kolay floresan gazetecilere takip edilebilir. Bizim durumumuzda (Şekil 2) Neurog3 4 etkinleştirmeden tamoksifen yokluğunda ve böylece Pdx1 promoteri (Pdx1-Ngn3-ER TM-nGFP) (Film 1) tarafından kontrol edilen bir nükleer GFP proteini eksprese eden transgenik bir fare kullanılmıştır. Organoid orta ile...

Watch this Scientific Journal Video about In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors at JoVE.com

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

Bu protokol açıklanan üç boyutlu kültür yöntemi dallı bir organ haline onların önemli genişleme, farklılaşma ve morfojenezinin dahil dağınık embriyonik fare pankreas atalarıdır, gelen pankreas gelişimini özetlediği. Bu yöntem, görüntüleme, niş fonksiyonel müdahale ve manipülasyon için uygun değildir.

Dağınık Fare Embriyonik progenitörlerin gelen Vitro Pankreas organogeneziste

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

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Biology

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

13.7K Görüntüleme.  Swiss Institute for Experimental Cancer Research.  Bu protokol açıklanan üç boyutlu kültür yöntemi dallı bir organ haline onların önemli genişleme, farklılaşma ve morfojenezinin dahil dağınık embriyonik fare pankreas atalarıdır, gelen pankreas gelişimini özetlediği. Bu yöntem, görüntüleme, niş fonksiyonel müdahale ve manipülasyon için uygun değildir. 

Video: In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

In Vitro Colony Tahliller Tri-güçlü Progenitör Hücreleri karakterize Yetişkin Fare Pankreas İzole

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Gelişim Biyolojisi

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 ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

Pluripotent Verimli Farklılaşma NKX6-1 için Kök Hücreler<sup&gt; +</sup&gt; Pankreas Progenitörler

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

İnsan Fetal Pankreas Hücre Kümeleri İzolasyon, Kültür ve Görüntüleme

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Tıp

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

İskele-free, üç boyutlu insülin ifade Pancreatoids fare pankreas ataları Vitro gelen nesil

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

Verimli üretimi, pankreas/oniki parmak bağırsağı Homeobox Protein 1+ Posterior Foregut/pankreas ataları hPSCs yapışma kültürlerde üzerinden

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

İnsan Pluripotent Kök Hücrelerinin 2D Kültür Sisteminde Pankreatik Beta Hücre Öncüllerine Farklılaşması

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

İnsülin ifade Koloni oluşturan atalarıdır için Kantitatif Testi

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Biyoloji

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.

İnsan Embriyonik endotel hücre Co-kültür aracılık Olgunlaşma Bir Yönetmen Farklılaşma Yaklaşım Pankreas insülin üreten hücreler için Kök Hücre

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Için bir sistem<em&gt; Ex vivo</emEmbriyonik Pankreas&gt; ekimi

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Dağınık Fare Embriyonik progenitörlerin gelen Vitro Pankreas organogeneziste

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