के लिए एक प्रणाली<em> पूर्व vivo</emभ्रूण अग्न्याशय> संवर्धन

Summary

यहाँ, हम अलगाव, संस्कृति और माउस भ्रूणीय अग्न्याशय के हेरफेर के लिए एक विधि का वर्णन है. यह एक उत्कृष्ट प्रतिनिधित्व करता है<em> पूर्व vivo</em> Morphogenesis भेदभाव, और अग्नाशय के विकास के विकास सहित विभिन्न पहलुओं, अध्ययन के लिए प्रणाली. अग्नाशय के कली explants कई दिनों के लिए संवर्धित किया जा सकता है और पूरे माउंट immunofluorescence और रहते इमेजिंग सहित विभिन्न अनुप्रयोगों, की एक श्रृंखला में प्रयोग किया जाता है.

Abstract

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 embryo^3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ^2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing β-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium^7,8. 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.

Protocol

यहाँ वर्णित प्रोटोकॉल मूल पेर्सिवल और सुस्त 9 में वर्णित और confocal माइक्रोस्कोपी के लिए अनुकूलित तकनीक से अनुकूलित किया गया है. 1. गिलास नीचे संस्कृति व्यंजन की कोटिंग निम्नलिखित…

Discussion

एक बार अग्नाशय भाग्य निर्दिष्ट किया जाता है, अग्नाशय पूर्वपुस्र्ष कोशिकाओं व्यापक प्रसार, भेदभाव और morphogenesis अंततः एक परिपक्व और कार्यात्मक ^2,4 अंग फार्म से गुजरना. वर्तमान में, कैसे शाखाओं में बंटी अग…

Materials

Name of the reagent	Company	Catalogue number	Comments
Antibodies: Carboxypeptidase E-cadherin F-actin Glucagon Insulin β1-integrin Pdx1 Pdx1 Phospho-Histone H3	AbD Serotec Invitrogen Molecular Probes ImmunoStar Millipore Millipore Abcam Hybridoma bank Cell Signalling	1810-0006 13-1900 A-12373 20076 4011-01 MAB1997 ab47267 F109-D12 9706
Basal Medium Eagle (BME)	Sigma	B1522-500ML	Kept in sterile conditions
Cell culture grade water	PAA	S15-012	Kept in sterile conditions
Culture dishes (glass-bottomed), 35-mm	MatTek Corporation	P35G-0-20-C
Donkey Serum	Chemicon	S30-100 ml
Fetal calf serum Gold	PAA	A15-151	Kept in sterile conditions
Fibronectin	Invitrogen	330100-8	Stock sol. 1 mg/ml in cell culture grade water
Gentamicin	Invitrogen	15750-037	Kept in sterile conditions
Glutamine	Invitrogen	25030-024	Kept in sterile conditions
4-well Multidishes	Nunc	176740
Microscopes: Inverted Confocal Microscope (LSM 700) Stereomicroscope (Discovery V12)	Zeiss Zeiss		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 μm) Transillumination from below and fiber-optic illumination from above
Paraformaldehyde	Roth	0335.3	Stock solution 20%
Pasteur Pipet (Glass), 150 mm	VWR	HECH567/1
Penicillin/Streptomycin	PAA	P11-010	Kept in sterile conditions
Petri dishes, 60 mm	Greiner Bio-One	628102
Petri dishes, 35 mm	Greiner Bio-One	627161
1X PBS, pH7.4	PAA	H15-002	Kept in sterile conditions
Spring Scissors 8 mm blade curved	Fine Science Tools	15023-10
Triton-X100	Roth	3051.3
Watchmaker’s foreceps Dumont #5	Roth	K342.1