Dissektion und Kultur der Maus Embryonale Niere
Summary
Dieses Protokoll beschreibt eine Methode zur Isolierung und Kultivierung von metanephrischen Rudimente aus Mäuseembryonen.
Abstract
Das Ziel dieses Protokolls ist es, eine Methode für die Sektion, Isolierung und Kultur der Maus metanephrischen Rudimente zu beschreiben.
Während der Säugetier-Nieren-Entwicklung, die beiden Vorläufer-Gewebe, die ureterische Knospe und das metanephrische Mesenchym, kommunizieren und wechselseitig zelluläre Mechanismen induzieren, um schließlich das Sammelsystem und die Nephrone der Niere zu bilden. Da Säugetier-Embryonen intrauterin wachsen und daher für den Beobachter unzugänglich sind, wurde eine Orgelkultur entwickelt. Mit dieser Methode ist es möglich, epithelial-mesenchymale Wechselwirkungen und zelluläres Verhalten während der Nierenorganogenese zu untersuchen. Darüber hinaus kann der Ursprung von angeborenen Nieren- und Urogenitaltrakt-Fehlbildungen untersucht werden. Nach sorgfältiger Dissektion werden die metanephrischen Rudimente auf einen Filter übertragen, der auf Kulturmedium schwimmt und in einem Zellkulturinkubator für mehrere Tage gehalten werden kann. Allerdings muss man sich bewusst sein, dass die Bedingungen sindKünstlich und könnte den Stoffwechsel im Gewebe beeinflussen. Auch konnte das Eindringen von Testsubstanzen aufgrund der extrazellulären Matrix und der im Explantat vorhandenen Basalmembran begrenzt werden.
Ein wesentlicher Vorteil der Orgelkultur ist, dass der Experimentator direkten Zugang zum Orgel haben kann. Diese Technologie ist billig, einfach und erlaubt eine Vielzahl von Modifikationen, wie die Zugabe von biologisch aktiven Substanzen, die Untersuchung von genetischen Varianten und die Anwendung fortschrittlicher bildgebender Verfahren.
Introduction
The mammalian kidney is derived from two primordial structures with mesodermal origin: the tubular epithelial ureteric bud and the metanephric mesenchyme. During nephrogenesis, the ureteric bud invades the metanephric mesenchyme and branches to form the collecting system. The metanephric mesenchyme gives rise to the epithelial elements of the nephrons. These processes occur in a precisely timed and spatially coordinated manner and are initiated by reciprocal inductive mechanisms. Both tissue components communicate and affect the other’s cell morphogenesis.
In the 1920s, it was Boyden who performed the in vivo obstruction of the mesonephric duct in chicken, providing the first indication of inductive interactions as separated nephric blastema fail to differentiate1. At about the same time, the first successful attempts to culture chicken nephric rudiments in a hanging drop were published. Subsequently, the organ culture was developed to study tissue interactions in mammalian organogenesis. In the 1950s, Grobstein developed a technique in which metanephric rudiments could be cultured on a filter. This technique was modified by Saxén, who placed the filter on a Trowell-type screen in a culture dish1. Over the years, many modifications and applications for organ culture have emerged. The method described here is based on Saxén’s technique but is simplified, as the filters float free on the medium and the diameter of the culture well only slightly exceeds the diameter of the filter, limiting unwanted movement of the filter.
Whole-organ culture is a classical, cheap, and simple but powerful tool to investigate cellular processes and intercellular communication during organogenesis. Organ culture allows for treatment with biological agents, such as growth factors, antibodies, antisense oligonucleotides, viruses, and peptides, as well as with pharmaceutical compounds and other chemicals. Also, gene function may be studied using explants derived from genetically modified mice or using inducible gene inactivation technology, such as the Cre-loxP system. This allows for the study of genetic mutations that cause embryonic lethality prior to the development of the kidney. Organ culture can also be combined with fluorescent tagging for gene function or lineage tracing and modern imaging techniques, which enable real-time monitoring of cell behavior2.
In the specific example provided here, the effect of EphrinB2-activated Eph-receptor signaling on the branching morphology of the ureteric bud was investigated. The morphology of the EphA4/EphB2 double-knockout mice suggested several severe defects in kidney development, which were detectable as early as embryonic day 11 (E11) and involved the ureteric bud, the ureter, and the common nephric duct3. Signaling via Eph receptors requires the clustering of the ligand-receptor dimer4. To over-activate Eph signaling, the kidney rudiments from E11.5 mouse embryos were cultured in the presence of clustered recombinant EphrinB2-Fc. EphrinB2 is a known ligand for the EphA4 receptor, which is expressed in the ureteric bud tips3.
Protocol
Representative Results
Discussion
Dieses Manuskript beschreibt eine Methode, um das sich entwickelnde metanephrische Anlagen aus dem Maus-Embryo zu isolieren und die Orgel-Rudimente zu kultivieren. Diese Methode ist eine Standardtechnik, wie sie von Grobstein 8 und Saxén 9 , 10 entwickelt wurde und von vielen anderen 11 , 12 angepasst und modifiziert wurde. Der Erfolg der Methode hängt hauptsächlich von der Dau…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Die Autoren danken Leif Oxburgh und Derek Adams für ihre Kenntnisse, Leif Oxburgh für die hilfreichen Kommentare zum Manuskript und Stefan Wölfl und Ulrike Müller für ihre technische Unterstützung und Saskia Schmitteckert, Julia Gobbert, Sascha Weyer und Viola Mayer für die Hilfe in der Labor. Diese Arbeit wurde von der Entwicklung, der Gesellschaft der Biologen (nach CP) unterstützt .
Materials
| DMEM/F-12 | Thermo Fisher Scientific | 21331020 | |
| Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher Scientific | 15140148 | |
| GlutaMAX Supplement | Thermo Fisher Scientific | 35050061 | |
| DPBS, calcium, magnesium | Thermo Fisher Scientific | 14040117 | use for dissection |
| holo-Transferrin human | Sigma-Aldrich | T0665 | |
| Insulin-Transferrin-Selenium (ITS -G) (100X) | Thermo Fisher Scientific | 41400045 | |
| Paraformaldehyde | Sigma-Aldrich | 158127 | |
| Amphotericin B solution | Sigma-Aldrich | A2942 | |
| Triton X-100 | Sigma-Aldrich | X100 | |
| Sodium azide | Sigma-Aldrich | S8032 | |
| Thimerosal | Sigma-Aldrich | T5125 | |
| Propyl gallate | Sigma-Aldrich | 2370 | |
| Mowiol 4-88 | Sigma-Aldrich | 81381 | |
| Glycerol | Sigma-Aldrich | G5516 | |
| Biotinylated Dolichorus Biflorus Agglutinin | Vector Laboratories | B-1035 | |
| Alexa488 conjugated Streptavidin | Jackson Immuno Research | 016-540-084 | |
| Recombinant Mouse Ephrin-B2 Fc Chimera Protein, CF | R&D Systems | 496-EB | |
| Recombinant Human IgG1 Fc, CF | R&D Systems | 110-HG-100 | |
| Goat Anti-Human IgG Fc Antibody | R&D Systems | G-102-C | |
| Phosphate buffered saline tablets | Sigma-Aldrich | P4417 | use for fixation and immunostaining |
| Dumont #5, biologie tips, INOX, 11cm |
agnthos.se | 0208-5-PS | 2 pairs of forceps are needed |
| Iris scissors, straight, 12cm | agnthos.se | 03-320-120 | |
| Dressing Forceps, straight, delicate, 13cm |
agnthos.se | 08-032-130 | |
| Petri dishes Nunclo Delta treated | Thermo Fisher Scientific | 150679 | |
| TMTP01300 Isopore Membrane Filter, polycarbonate, Hydrophilic, 5.0 µm, 13 mm, white, plain | MerckMillipore | TMTP01300 | |
| Nunclon Multidishes 4 wells, flat bottom |
Sigma-Aldrich | D6789-1CS | |
| Microscope cover glass24x50mm thickn. No.1.5H 0.17+/-0.005mm | nordicbiolabs | 107222 | |
| Cover glasses No.1.5, 18x18mm | nordicbiolabs | 102032 | |
| Slides ~76x26x1, 1/2-w. ground plain | nordicbiolabs | 1030418 | |
| VWR Razor Blades | VWR | 55411-055 | |
| 50 mL centrifuge tubes | Sigma-Aldrich | CLS430828 | |
| 15 mL centrifuge tubes | Sigma-Aldrich | CLS430055 | |
| Whatman prepleated qualitative filter paper, Grade 113V, creped | Sigma-Aldrich | WHA1213125 | |
| Fixed stage research mircoscope | Olympus | BX61WI | |
| Black 6 inbred mice, male, C57BL/6NTac | Taconic | B6-M | |
| Black 6 inbred mice,female, C57BL/6NTac | Taconic | B6-F | |
| Greenough Stereo Microscope | Leica | Leica S6 E |
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