Name: generation of scaffold-free, three-dimensional insulin expressing pancreatoids from mouse pancreatic progenitors in vitro
Uploaded: 2018-06-02T14:00:00
Description: Watch this Scientific Journal Video about Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro at JoVE.com

हम सहायक प्रोटोकॉल और पांडुलिपि के बारे में चर्चा के लिए Jolanta Chmielowiec धंयवाद । हम भी फोकल माइक्रोस्कोप के लिए उपयोग के लिए बिंयामीन Arenkiel धंयवाद । इस कार्य को NIH (P30-DK079638 को M.B. और T32HL092332-13 को M.A.S. और M.B.), McNair मेडिकल फाउंडेशन (M.B. को), और फोकल कोर को बीसीएम बौद्धिक और विकासात्मक विकलांग अनुसंधान केंद्र (NIH U54 HD083092 से Eunice) द्वारा समर्थित किया गया कैनेडी श्राइवर राष्ट्रीय बाल स्वास्थ्य और मानव विकास संस्थान) ।

इस पद्धति में वर्णित सभी पशु प्रयोगों को Baylor कॉलेज ऑफ मेडिसिन की संस्थागत एनिमल केयर एंड फीमेल कमेटी ने अनुमोदित किया था ।
1. माउस भ्रूण दिवस की तैयारी १०.५ अग्नाशय Progenitors
नोट: इस प्रोटो?...

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सेल संस्कृति मॉडल की प्रगति ठीक से मॉडल विकास के लिए महत्वपूर्ण है, चिकित्सकीय प्रासंगिक सेल प्रकार, परीक्षण दवा प्रभावकारिता, या यहां तक कि रोगियों के लिए प्रत्यारोपण का उत्पादन । हालांकि, कृत्रिम रू...

Delineating सामांय विकास और शरीर विज्ञान के तंत्र को रोग एटियलजि समझ सर्वोपरि है और अंततः उपचार विधियों की खेती । जबकि संवर्धन और अंतर स्टेम कोशिकाओं के विकास के त्वरित और उच्च प्रवाह विश्लेषण में सक्षम बनाता है, यह ज्ञान के मौजूदा शरीर सेल भाग्य विनियमन और कृत्रिम रूप से एक अपेक्षाकृत में recapitulates विकास तंत्र के बारे में द्वारा सीमित है समरूप, दो आयामी स्थिति1,2। न केवल vivo बाह्य प्रभाव से प्रभावित विकास में है, आला और organogenesis गाइड करने के लिए paracrine संकेतों और संगठनात्मक समर्थन प्रदान वातावरण में विभिंन प्रकार के सेल के साथ, लेकिन इन कोशिकाओं के समारोह में भी उनके पर निर्भर करता है मार्गदर्शन के लिए परिवेश3,4,5। इन बाहरी संकेतों के महत्व को देखते हुए, विभेदक प्रोटोकॉल की सीमाओं, और vivo माउस मॉडल में की श्रमसाध्य प्रकृति, नई प्रणालियों के लिए प्रयोगात्मक विकास प्रक्रियाओं और फिजियोलॉजी की जांच करने के लिए आवश्यक हैं ।
प्रोटोकॉल के उद्भव के लिए तीन उत्पंन आयामी, जटिल organoids एक सुविधाजनक और अनुकूल प्रणाली का अध्ययन करने के लिए organogenesis, फिजियोलॉजी, दवा प्रभावकारिता प्रदान करता है, और यहां तक कि रोगजनन। विभिंन प्रणालियों के लिए murine organoids की स्थापना जैसे पेट6 और आंत7 organogenesis की हमारी समझ का विस्तार किया है, vivo में से कम प्रतिबंधों के साथ विकासात्मक जटिलताओं का अध्ययन करने के लिए एक उपकरण प्रदान और इन विट्रो मॉडल । murine organoid गठन और मानव pluripotent स्टेम कोशिकाओं के आगमन में इन अग्रिमों के कारण, मानव आंत्र8, रेटिना9, गुर्दे10,11, और सेरेब्रल12 organoids का उत्पादन किया गया है, और इस प्रदर्शनों की अध्यक्षता केवल मौजूदा विकास के तंत्र के बारे में ज्ञान द्वारा सीमित है ।
विशेष रुचि के अग्नाशय organoids की पीढ़ी है, रोगों के असंख्य के रूप में कोष्ठकी कोशिकाओं और नलिकाओं exocrine में अग्नाशय कमी13, अग्नाशयशोथ में कोष्ठकी कोशिकाओं सहित अलग अग्नाशय कोशिका प्रकार, प्लेग14, और डायबिटीज में बीटा कोशिकाएं15. इन विभिंन प्रकार के सेल के विकास के बारे में ज्ञान प्राप्त उनकी विकृति को समझने में सहायता कर सकता है और कर सकते हैं, भी, व्यक्तिगत दवा स्क्रीनिंग या प्रत्यारोपण के लिए एक मंच के रूप में कार्य । इससे पहले, Greggio एट अल. murine अग्नाशय organoids बनाने के लिए एक विधि विकसित की है कि vivo morphogenesis में दोहराऊंगा और संगठित, तीन आयामी, जटिल संरचनाओं सभी प्रमुख अग्नाशय उपकला कोशिका से बना विकसित 16,17प्रकार । यह अग्नाशय के क्षेत्र में एक प्रमुख कदम आगे है, विशेष रूप से इन विट्रो में कोशिकाओं बनाने बीटा सेल विकास की जैविक जांच सक्षम कर सकते हैं । हालांकि, अंत में इस प्रोटोकॉल में गठित कोशिकाओं की कमी जब तक organoids ऊतक में प्रत्यारोपित किया गया, जहां आला बातचीत और अनुदेशात्मक cues17प्रदान कर सकता है । mesenchyme आला के सबसे बड़े हिस्से का गठन किया, भारी organogenesis के प्रारंभिक दौर से विकासशील उपकला ढंकना अंत में स्रावी फाड़ना और भेदभाव3सहित बाद के चरणों के लिए,4, 18. विकासशील अग्ंयाशय के साथ mesenchyme की बातचीत अभी तक बाह्य संकेतन और organogenesis का अध्ययन करने के लिए vivo सेलुलर जटिलता में बनाए रखने के महत्व का एक और उदाहरण है ।
यहां, हम का वर्णन कैसे तीन उत्पंन करने के लिए आयामी अग्नाशय organoids, pancreatoids, असंबद्ध e 10.5 murine अग्नाशय progenitors से, शब्द । इन pancreatoids को बनाए रखने के मूल mesenchyme, स्व-मुक्त अस्थायी स्थितियों में इकट्ठा, और अंत: स्रावी बीटा कोशिकाओं की एक मजबूत संख्या सहित सभी प्रमुख अग्नाशय कोशिका प्रकार, उत्पन्न19. यह दृष्टिकोण सबसे अच्छा अंत में स्रावी विकास के विश्लेषण के लिए अनुकूल है, पिछले प्रोटोकॉल के रूप में मजबूत अंत में अंतर विभेदक कमी. हालांकि, Greggio एट अल द्वारा वर्णित के रूप में अग्नाशय organoids के लिए प्रोटोकॉल का उपयोग कर. बेहतर अग्नाशय उपकला शाखाओं और morphogenesis के विश्लेषण के लिए अनुकूल है, के रूप में शाखाओं में अधिक pancreatoids में सीमित है ।

<table border="0" cellpadding="0" cellspacing="0" style="width:849px;" width="849">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">2-Mercaptoethanol</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">M6250</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Aspirator Tube Assemblies for Calibrated Microcapillary Pipettes</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">A5177</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BarnStead NanoPure Nuclease-free water</td>
			<td>ThermoFisher</td>
			<td>D119</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Borosilicate Capillary Tubes</td>
			<td style="width:123px;">Sutter Instruments</td>
			<td style="width:344px;">GB1007515</td>
			<td style="width:167px;">O.D. 1mm, I.D. 0.75mm, 1.5cm length</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">CaCl2</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">C5080</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Cell-Repellent 96-Well Microplate</td>
			<td style="width:123px;">Greiner Bio-One</td>
			<td style="width:344px;">650970</td>
			<td style="width:167px;">U-bottom</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Centrifuge 5424 R</td>
			<td>Eppendorf</td>
			<td>5401000013</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Chloroform</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">233306</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Chromogranin-A antibody</td>
			<td>Abcam</td>
			<td>ab15160</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Compact, Modular Stereo Microscope M60</td>
			<td style="width:123px;">Leica</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Countess Automated Cell Counter</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">C10310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Countess Cell Counter Slides</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">C10312</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CryoStar NX70</td>
			<td>ThermoFisher</td>
			<td>957000L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">D-(+)-Glucose</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">G7528</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">DAPI (4&#39;,6-Diamidine-2&#39;-phenylindole-dihydrochloride)</td>
			<td style="width:123px;">Roche</td>
			<td style="width:344px;">10 236 276 001</td>
			<td style="width:167px;">Powder</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DBA antibody</td>
			<td>Vector Lab</td>
			<td>RL-1032</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dispase II, Powder</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:344px;">17105041</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">DMEM/F-12, HEPES</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:344px;">11330032</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dnase I</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">18068-015</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dumont #5 Forceps</td>
			<td style="width:123px;">Fine Science Tools</td>
			<td style="width:344px;">11251-10</td>
			<td style="width:167px;">0.05 x 0.02 mm; Titanium; Biology tip</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">EGF (Epidermal growth factor)</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">E9644</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Ethanol, 200 Proof</td>
			<td style="width:123px;">Decon Laboratories</td>
			<td style="width:344px;">2716</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Forma Steri Cycle CO2 Incubators</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:344px;">370</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Fluoromount-G</td>
			<td style="width:123px;">Southern Biotech</td>
			<td style="width:344px;">OB10001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Heparin sodium salt from porcine intestinal mucosa</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">H3149-10KU</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">INSM1 Antibody</td>
			<td style="width:123px;">Santa Cruz BioTechnology</td>
			<td style="width:344px;">sc-271408</td>
			<td style="width:167px;">Polyclonal Mouse IgG</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Isopropanol</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:344px;">a4164</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isothesia Isoflurane, USP</td>
			<td>Henry Schein</td>
			<td>11695-6776-2</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Insulin Antibody</td>
			<td style="width:123px;">Dako</td>
			<td style="width:344px;">A056401</td>
			<td style="width:167px;">Polyclonal Guinea Pig</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">KAPA SYBR FAST Universal</td>
			<td style="width:123px;">KAPA Biosystems</td>
			<td style="width:344px;">KK4618</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">KCl</td>
			<td style="width:123px;">KaryoMax</td>
			<td>10575090</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">KnockOut Serum Replacement</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">10828028</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Leica TCS SPE High-Resolution Spectral Confocal</td>
			<td style="width:123px;">Leica</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">MgCl2</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">442615</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Mouse C-Peptide ELISA</td>
			<td style="width:123px;">ALPCO</td>
			<td style="width:344px;">80-CPTMS-E01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Mouse Ultrasensitive Insulin ELISA</td>
			<td style="width:123px;">ALPCO</td>
			<td style="width:344px;">80-INSMSU-E01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">MX35 Microtome Blades</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:344px;">3052835</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">NaCl</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">S7653</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">NaHCO3</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">S3817</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">NaH2PO4</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Normal Donkey Serum</td>
			<td style="width:123px;">Jackson Immuno Research</td>
			<td style="width:344px;">017-000-121</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Paraformaldehyde</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">158127</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">PBS 1X</td>
			<td style="width:123px;">Corning</td>
			<td style="width:344px;">21-040-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pdx1 antibody</td>
			<td>DSHB</td>
			<td>F6A11</td>
			<td>Monoclonal Mouse MIgG1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Peel-A-Way Disposable Embedding Molds</td>
			<td style="width:123px;">VWR</td>
			<td style="width:344px;">15160-157</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Penicillin-Streptomycin Solution</td>
			<td style="width:123px;">Corning</td>
			<td style="width:344px;">MT30002CI</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">PMA (Phorbol 12-Myristate 13-Acetate)</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">P1585</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Protein LoBind Microcentrifuge Tubes</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:344px;">22431081</td>
			<td style="width:167px;">1.5mL Capacity</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Recombinant Human FGF-10 Protein</td>
			<td style="width:123px;">R&#38;D Systems</td>
			<td style="width:344px;">345-FG</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Recombinant Human FGF-Acidic</td>
			<td style="width:123px;">Peprotech</td>
			<td style="width:344px;">100-17A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Recombinant Human R-Spondin I Protein</td>
			<td style="width:123px;">R&#38;D Systems</td>
			<td style="width:344px;">4546-RS</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">BenchRocker 2D</td>
			<td style="width:123px;">Benchmark</td>
			<td style="width:344px;">BR2000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sucrose 500g</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">S0389</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">SuperFrost Plus Microscope Slides</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:344px;">12-550-15</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Super Pap Pen</td>
			<td style="width:123px;">Electron Microscopy Sciences</td>
			<td style="width:344px;">71310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Thermomixer R</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:344px;">05-412-401</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Tissue Tek O.C.T. Compound</td>
			<td style="width:123px;">Sakura</td>
			<td style="width:344px;">4583</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Triton X-100</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">T8787</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">TRIzol Reagent</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">15596018</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">TrypLE Express</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">12604039</td>
			<td style="width:167px;">(1x), no Phenol Red</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Trypan Blue Stain</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">15250061</td>
			<td style="width:167px;">For cell counting slides</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Trypsin-EDTA (0.05%)</td>
			<td style="width:123px;">Corning</td>
			<td style="width:344px;">25-052-CI</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:123px;">Gibco</td>
			<td>25200072</td>
			<td style="width:167px;">Phenol Red</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Ultra-Low Attachment 24-Well Plate</td>
			<td style="width:123px;">Corning</td>
			<td>3473</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Ultra-Low Attachment Spheroid Plate 96-Well</td>
			<td style="width:123px;">Corning</td>
			<td>4520</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Vimentin Antibody</td>
			<td style="width:123px;">EMD Millipore</td>
			<td style="width:344px;">AB5733</td>
			<td style="width:167px;">Polyclonal Chicken IgY</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Vortex Genie</td>
			<td style="width:123px;">BioExpres</td>
			<td style="width:344px;">S-7350-1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Y-27632 Dihydrochloride</td>
			<td style="width:123px;">R&#38;D Systems</td>
			<td>1254</td>
			<td style="width:167px;">Also known as ROCK inhibitor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Zeiss 710 Confocal Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of scaffold-free, three-dimensional insulin expressing pancreatoids from mouse pancreatic progenitors in vitro

अग्ंयाशय एक जटिल कई विभिंन प्रकार के सेल है कि एक साथ काम करने के लिए रक्त ग्लूकोज homeostasis और पाचन को विनियमित अंग बना है । इन सेल प्रकार एंजाइम स्रावित कोष्ठकी कोशिकाओं, पेट के लिए एंजाइमों की ढुलाई के लिए जिंमेदार एक arborized डक्टर प्रणाली, और हार्मोन का उत्पादन अंत में स्रावी कोशिकाओं शामिल हैं ।
अंत-स्रावी बीटा-कोशिकाओं शरीर में एकमात्र कोशिका प्रकार है कि रक्त शर्करा की मात्रा कम करने के लिए इंसुलिन का उत्पादन कर रहे हैं । मधुमेह, एक नुकसान या बीटा कोशिकाओं की शिथिलता की विशेषता रोग, महामारी अनुपात तक पहुंच रहा है । इस प्रकार, यह आवश्यक है कि बीटा सेल विकास की जांच करने के लिए प्रोटोकॉल स्थापित करने के लिए इस्तेमाल किया जा सकता है स्क्रीनिंग प्रयोजनों के लिए दवा और कोशिका आधारित चिकित्सकीय प्राप्त । हालांकि माउस के विकास की प्रायोगिक जांच जरूरी है, vivo में पढ़ाई श्रमसाध्य और समय लेने वाली है. कल्चरल सेल स्क्रीनिंग के लिए एक अधिक सुविधाजनक मंच प्रदान करते हैं; हालांकि, वे सेलुलर विविधता, वास्तु संगठन और सेलुलर बातचीत को बनाए रखने में असमर्थ है vivo में पाया । इस प्रकार, यह नए उपकरणों के विकास के लिए आवश्यक है अग्नाशय organogenesis और फिजियोलॉजी की जांच ।
अग्नाशय उपकला कोशिकाओं के रूप में कोशिकाओं को संगठित और जटिल, शारीरिक रूप से सक्षम वयस्क अंग में अंतर organogenesis की शुरुआत से mesenchyme के साथ निकट सहयोग में विकसित. अग्नाशय mesenchyme अंत में स्रावी विकास के लिए महत्वपूर्ण संकेत प्रदान करता है, जिनमें से कई अच्छी तरह से अभी तक समझ में नहीं हैं, इस प्रकार के दौरान दोहराऊंगा करने के लिए मुश्किल इन विट्रो संस्कृति. यहां, हम संस्कृति के लिए एक प्रोटोकॉल का वर्णन तीन आयामी, सेलुलर जटिल माउस organoids कि mesenchyme बनाए रखने, pancreatoids । ई 10.5 murine अग्नाशय कली, असंबद्ध, और एक पाड़ मुक्त वातावरण में संस्कृति विदारक है । इन अस्थायी कोशिकाओं mesenchyme विकासशील pancreatoid और अंत में कोष्ठकी और वाहिनी कोशिकाओं के साथ विकासशील-स्रावी बीटा कोशिकाओं की एक मजबूत संख्या को घेर के साथ इकट्ठा स्व. इस प्रणाली के लिए सेल भाग्य निर्धारण, संरचनात्मक संगठन, और morphogenesis, सेल सेल बातचीत organogenesis के दौरान, या दवा, छोटे अणु, या आनुवंशिक स्क्रीनिंग के लिए अध्ययन करने के लिए इस्तेमाल किया जा सकता है ।

सावधान माउस भ्रूण के गर्भाशय के हॉर्न से 10.5 ई में विच्छेदन आगे विच्छेदन के लिए पंजाब में नुकसान हुआ भ्रूण उपज (चित्रा 1ए) चाहिए । जठरांत्र संबंधी मार्ग कुशलतापूर्वक भ्रूण स...

Watch this Scientific Journal Video about Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro at JoVE.com

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

पाड़-मुक्त, तीन आयामी इंसुलिन व्यक्त Pancreatoids से माउस अग्नाशय Progenitors इन विट्रो में की पीढ़ी

यहां, हम एक प्रोटोकॉल पेश करने के लिए इंसुलिन व्यक्त 3 डी murine pancreatoids मुक्त-फ़्लोटिंग ई 10.5 असंबद्ध अग्नाशय progenitors और एसोसिएटेड mesenchyme से उत्पंन ।

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

This method could help answer key questions in the pancreatic field by using these pancreatoids to understand how endocrine cells form in an architecturally and cellularly complex environment. The main advantage of this technique is that the pancreatoids develop endocrine cells due to the inclusion of the surrounding mesenchyme. Generally, individuals new to this method will struggle because the pancreas is very small at e10.5.And the dissection is crucial to the formation of pancreatoids to get enough non-contaminating tissue. To set up for dissection, fill an ice bucket and place a container of phosphate buffered saline in the ice. Clean two fine-tip forceps and dissection scissors with 70%ethanol.Near the dissection area, set a container with at least three borosilicate capillary tubes, a lighter, and filtered 1000 microliter pipet tips. Next, pipet at least one milliliter of 1.25 milligram per milliliter cold dispase diluted in sterilized water in the second well of a 12-well plate on ice. Pipet PBS in the first, third, and fourth wells of the 12-well plate.Also, pipet one milliliter of 0.05%trypsin in a 1.5 milliliter tube on ice. After euthanizing e10.5 timed pregnant mice in accordance with IACUC guidelines, spray the abdomen with 70%ethanol to clean the region. Then, make a V-shaped incision at the genital area of the mouse using scissors and forceps and continue the incision up to the diaphragm.Carefully excise the uterus by making an incision at the uppermost lateral portions of the uterus, pulling the organ upwards away from the mouse and following this incision down to the genital region before repeating on the opposite side. Place the excised uterus in a 10 centimeter petri dish with the cold PBS. Under a light dissection microscope, place the petri dish and carefully open the uterine tissue by placing two forceps between each embryo and peeling tissue away from the yolk sac.Transfer the embryos by grasping the yolk sac gently with forceps and placing them in a 10 centimeter petri dish with fresh, cold PBS. Place the petri dish on ice. Next, place multiple PBS drops on the 10 centimeter petri dish lid.Use these droplets to transfer the embryo during dissection as extra-embryonic tissues are removed to ensure visibility. Transfer an embryo to a PBS drop and gently remove it from the yolk sac using forceps while the remaining embryos stay in PBS on ice. Remove the head before moving the embryo to a new PBS drop using forceps to lightly scoop the embryo without damaging the tissue.In a new PBS drop, remove the four limb buds using forceps. Place the forceps into the opening where limb bud was present and gently tear only the most external tissue anteriorly. Rotate the embryo and repeat in the posterior direction, stopping at the hind limb bud.At this point, the gastrointestinal tract should be visible. The posterior region of the gastrointestinal tract has a slight bend;insert forceps behind this bend in the opening between the gastrointestinal tract and the spinal region at the body wall. Detach the gastrointestinal tract, working slowly upwards until the most anterior region is reached, where the cardiac region connects.In the following steps, it is critical to avoid the surrounding tissue in the culture, so as to not contaminate the pancreatoids with non-pancreatic tissue. However, if you do not grab the whole pancreatic bud, then the number and size of the pancreatoids will be impaired. Transfer the gastrointestinal tract to the fresh PBS droplet.Continue to remove the outer tissue surrounding the gastrointestinal tract. Once this outer tissue is removed, the pancreatic bud can be visualized posterior to the liver buds, in between the stomach and intestine. Then, take forceps and gently pinch the tissue beneath the protruding bud in the intestine and slightly lift the pancreatic bud off.After pinching the region where the bud connects to the intestine, the bud is separated. Wash one time in a new PBS bubble before transferring the bud into the first well of the 12-well plate and cold PBS on ice. Repeat until all of the buds are collected from the embryos and placed in the first well of the 12-well plate.Next, place the 12 well dish under the light dissection microscope. Count the number of pancreatic buds successfully dissected to later calculate the split ratio. Place a filtered 1000 microliter pipet tip into the pipet with the capillary tube attached.Flame sterilize the capillary tube and create a bend in the tube for the ease of use. Use the capillary to transfer the buds to the second well containing cold dispase solution for two minutes before transferring to the third well containing clean PBS. Pipet the buds up and down and transfer them to the fourth well containing clean PBS.Finally, using the capillary device, transfer the buds to the 1.5 milliliter tube with 0.05%trypsin. Place the tube in the pre-warmed 37 degrees celsius shaker and shake at 1, 500 RPM for four to five minutes. After vortexing for approximately 10 seconds, immediately place the tube in the centrifuge and spin for five minutes at 200g.Cautiously remove all but approximately 50 microliters of solution as to not disturb the centrifuged cells. For every bud collected at 400 microliters of organogenesis media to the centrifuged cells, pulse vortex three times and plate 100 microliters per well of a low-attachment 96 well plate, splitting buds at a one to four ratio. Check under the microscope to visualize dissociated cells freely floating.Then place the culture dish in a 37 degrees celsius incubator with 5%carbon dioxide on a rocker at medium speed. Careful dissection of mouse embryos at e10.5 from the uterine horn should yield undamaged embryos in PBS for further dissection. The gastrointestinal tract can be efficiently removed from the embryo, permitting discernment of the dorsal pancreatic bud at the junction of the intestine and stomach.In organogenesis media, these free-floating scaffold-free cells self-assemble and organize into three-dimensional pancreatoids that grow and persist for at least 10 days in culture. The application of a protein kinase C activator at high concentrations alters the morphology of developing pancreatoids, leading to loosely associated epithelial cells and increased branching. Additionally, the use of transgenic animals allows visualization of cells as they differentiate in real time.To assess the association of pancreatic mesenchyme tissue with pancreatic epithelial cells, immunostaining can be performed for markers of each tissue. Immunostaining of a duct marker with an endocrine marker and nuclei, reveal multi-lineage formation in pancreatoids. A mesenchyme marker co-stained with a pancreatic progenitor marker shows that the mesenchyme envelops the pancreatoid.Beta cell development is revealed by staining for the epithelial marker and the beta cell marker. Using quantitative PCR analysis, transcripts of progenitors and differentiating cells can be assessed, such as progenitor genes and differentiated markers. Once mastered, this technique can be done in approximately one hour, including prep time, if it is performed properly.After watching this video, you should have a good understanding of how to perform the dissection to obtain pancreatic tissue from 10.5 mice to generate pancreatoids. While attempting this procedure, it's important to remember to carefully remove the GI tract from the embryos, otherwise it will be very difficult to find the pancreatic bud. Following this procedure, other methods like GSIS and ELISA can be performed in order to answer additional questions, like how much insulin or C-peptide is produced after glucose stimulation.After its development, this technique paved the way for us to explore the morphological consequences of PKC activation using small molecules. A way of further implementing this system to study the effect of transcription factor of expression and even to perform important epistasis experiments.

Research

JoVE Journal

Developmental Biology

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

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Thymic की विशेषता का उपयोग कर माउस भ्रूण में progenitors निपटाने<em&gt; Vivo</em&gt; और<em&gt; इन विट्रो</em&gt; Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

मानव प्रेरित स्टेम कोशिकाओं से व्युत्पन्न पिरामिड न्यूरॉन्स से वृक्ष के समान spines के तीन आयामी मात्रा

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

मानव Pluripotent स्टेम सेल से आदिम और निश्चित टेम Progenitors के विभेद का निर्देशन

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

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

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

Pluripotent के कुशल भेदभाव स्टेम कोशिकाओं को NKX6-1<sup&gt; +</sup&gt; अग्नाशय प्रोजेनिटर्स

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

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

दो और तीन आयामी embryoid निकायों में मूषक भ्रूणीय स्टेम कोशिकाओं की रेटिनोइक एसिड प्रेरित तंत्रिका विभेदन का विश्लेषण

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

माउस में प्रारंभिक गर्भावस्था लक्षण वर्णन के लिए तीन आयामी उच्च आवृत्ति ट्रा का एक उपंयास का उपयोग

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

तीन आयामी भड़काऊ मानव ऊतक Gingiva के समकक्ष

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

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

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

अग्ंयाशय के कुशल पीढ़ी/ग्रहणी Homeobox प्रोटीन 1+ पश् चवर्ती अग्रगुट/अग्ंयाशय आसंजन संस्कृतियों में hPSCs से Progenitors

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

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

हृदय Progenitors और वेंट्रिकुलर की तरह पहले दिल क्षेत्र की पीढ़ी-मानव Pluripotent स्टेम कोशिकाओं से Cardiomyocytes की तरह

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

मानव प्रेरित Pluripotent स्टेम सेल से Somite डेरिवेटिव के विट्रो पीढ़ी में

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...