Name: a system for ex vivo culturing of embryonic pancreas
Uploaded: 2012-08-27T12:00:00
Description: Watch this Scientific Journal Video about A System for ex vivo Culturing of Embryonic Pancreas at JoVE.com

Research in the Spagnoli lab. is funded by the Helmholtz Association, FP7-IRG-2008-ENDOPANC grant and ERC-2009-Starting HEPATOPANCREATIC Grant.

The protocol described here has been adapted from the technique originally described in Percival and Slack9 and optimized for confocal microscopy.
1. Coating of Glass Bottom Culture Dishes
The following steps should be carried out under sterile conditions in a laminar flow hood.
<ol>
 <li>Pancreatic explants are cultured in 35-mm Petri dishes with 20-mm diameter glass microwell bottom (e.g. MatTek Corporation). Use one glass bottom dish per exp...

<ol>
	<li>Slack, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+biology+of+the+pancreas.">Developmental biology of the pancreas.</a> Development. 121, 1569-1580 (1995).</li><li>Pan, F., Wright, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreas+organogenesis:+from+bud+to+plexus+to+gland.">Pancreas organogenesis: from bud to plexus to gland.</a> Dev. Dyn. 240, 530-565 (2011).</li><li>Puri, S., Hebrok, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Plasticity+within+the+Pancreas-+Lessons+Learned+from+Development.">Cellular Plasticity within the Pancreas- Lessons Learned from Development.</a> Developmental Cell. 18, 342-356 (2010).</li><li>Spagnoli, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+endoderm+to+pancreas:+a+multistep+journey.">From endoderm to pancreas: a multistep journey.</a> Cell. Mol. Life Sci. 64, 2378-2390 (2007).</li><li>Hick, A. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+primitive+duct+formation+in+the+pancreas+and+submandibular+glands:+a+role+for+SDF-1.">Mechanism of primitive duct formation in the pancreas and submandibular glands: a role for SDF-1.</a> BMC Dev. Biol. 9, 1-17 (2009).</li><li>Villasenor, A., Chong, D., Henkemeyer, M., Cleaver, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+dynamics+of+pancreatic+branching+morphogenesis.">Epithelial dynamics of pancreatic branching morphogenesis.</a> Development. 137, 4295-4305 (2010).</li><li>Kesavan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc42-Mediated+Tubulogenesis+Controls+Cell+Specification.">Cdc42-Mediated Tubulogenesis Controls Cell Specification.</a> Cell. 139, 791-801 (2009).</li><li>Zhou, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Multipotent+Progenitor+Domain+Guides+Pancreatic+Organogenesis.">A Multipotent Progenitor Domain Guides Pancreatic Organogenesis.</a> Developmental Cell. 13, 103-114 (2007).</li><li>Percival, A., Slack, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+pancreatic+development+using+a+cell+lineage+label.">Analysis of pancreatic development using a cell lineage label.</a> Exp. Cell Res. 247, 123-132 (1999).</li><li>Miralles, F., Czernichow, P., Ozaki, K., Itoh, N., Scharfmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+through+fibroblast+growth+factor+receptor+2b+plays+a+key+role+in+the+development+of+the+exocrine+pancreas.">Signaling through fibroblast growth factor receptor 2b plays a key role in the development of the exocrine pancreas.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 6267-6272 (1999).</li><li>Puri, S., Hebrok, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+embryonic+pancreas+development+using+real-time+imaging.">Dynamics of embryonic pancreas development using real-time imaging.</a> Dev. Biol. 306, 82-93 (2007).</li><li>Magenheim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessels+restrain+pancreas+branching,+differentiation+and+growth.">Blood vessels restrain pancreas branching, differentiation and growth.</a> Development. 138, 4743-4752 (2011).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: A Laboratory Manual. , (2003).</li><li>Horb, L. D., Slack, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cell+division+in+branching+morphogenesis+and+differentiation+of+the+embryonic+pancreas.">Role of cell division in branching morphogenesis and differentiation of the embryonic pancreas.</a> Int. J. Dev. Biol. 44, 791-796 (2000).</li><li>Muzumdar, M., 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>

Once pancreatic fate is specified, pancreatic progenitor cells undergo extensive proliferation, differentiation and morphogenesis to eventually form a mature and functional organ2,4. At present, how branching takes place in the pancreas and how it is connected to progenitor proliferation and differentiation is largely unknown. Pancreatic explant cultures represent an ideal system to elucidate these processes ex vivo5,9,11. By combining live-cell imaging with ex vivo explants...

<table border="1">
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Antibodies: Carboxypeptidase E-cadherin F-actin Glucagon Insulin &beta;1-integrin Pdx1 Pdx1 Phospho-Histone H3</td>
 <td>AbD Serotec Invitrogen Molecular Probes ImmunoStar Millipore Millipore Abcam Hybridoma bank Cell Signalling</td>
 <td>1810-0006 13-1900 A-12373 20076 4011-01 MAB1997 ab47267 F109-D12 9706</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Basal Medium Eagle (BME)</td>
 <td>Sigma</td>
 <td>B1522-500ML</td>
 <td>Kept in sterile conditions</td>
 </tr>
 <tr>
 <td>Cell culture grade water</td>
 <td>PAA</td>
 <td>S15-012</td>
 <td>Kept in sterile conditions</td>
 </tr>
 <tr>
 <td>Culture dishes (glass-bottomed), 35-mm </td>
 <td>MatTek Corporation </td>
 <td>P35G-0-20-C </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Donkey Serum</td>
 <td>Chemicon</td>
 <td>S30-100 ml</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal calf serum Gold</td>
 <td>PAA</td>
 <td>A15-151</td>
 <td>Kept in sterile conditions</td>
 </tr>
 <tr>
 <td>Fibronectin</td>
 <td>Invitrogen</td>
 <td>330100-8</td>
 <td>Stock sol. 1 mg/ml in cell culture grade water</td>
 </tr>
 <tr>
 <td>Gentamicin </td>
 <td>Invitrogen</td>
 <td>15750-037</td>
 <td>Kept in sterile conditions</td>
 </tr>
 <tr>
 <td>Glutamine</td>
 <td>Invitrogen</td>
 <td>25030-024</td>
 <td>Kept in sterile conditions</td>
 </tr>
 <tr>
 <td>4-well Multidishes</td>
 <td>Nunc</td>
 <td>176740</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microscopes: Inverted Confocal Microscope (LSM 700) Stereomicroscope (Discovery V12)</td>
 <td>Zeiss Zeiss</td>
 <td>&nbsp;</td>
 <td>Objectives: C-Apochromat 10X / 0.45 W M27 (work. dist. 1.8 mm; imaging depth ~100 mm); C-Apochromat 40X / 1.2 W Corr M27 (work. dist. 0.28 mm; ~imaging depth 50 &mu;m) Transillumination from below and fiber-optic illumination from above</td>
 </tr>
 <tr>
 <td>Paraformaldehyde</td>
 <td>Roth</td>
 <td>0335.3</td>
 <td>Stock solution 20% </td>
 </tr>
 <tr>
 <td>Pasteur Pipet (Glass), 150 mm</td>
 <td>VWR</td>
 <td>HECH567/1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin</td>
 <td>PAA</td>
 <td>P11-010</td>
 <td>Kept in sterile conditions</td>
 </tr>
 <tr>
 <td>Petri dishes, 60 mm</td>
 <td>Greiner Bio-One</td>
 <td>628102</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Petri dishes, 35 mm</td>
 <td>Greiner Bio-One</td>
 <td>627161</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1X PBS, pH7.4</td>
 <td>PAA</td>
 <td>H15-002</td>
 <td>Kept in sterile conditions</td>
 </tr>
 <tr>
 <td>Spring Scissors 8 mm blade curved</td>
 <td>Fine Science Tools</td>
 <td>15023-10</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Triton-X100</td>
 <td>Roth</td>
 <td>3051.3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Watchmaker's foreceps Dumont #5</td>
 <td>Roth</td>
 <td>K342.1</td>
 <td>&nbsp;</td>
 </tr>
 </table>

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.

Watch this Scientific Journal Video about A System for ex vivo Culturing of Embryonic Pancreas at JoVE.com

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.

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 ...

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