Summary
Denne protokol beskriver en metode til isolering og dyrkning af metanephriske rudimenter fra museembryoner.
Abstract
Formålet med denne protokol er at beskrive en metode til dissektion, isolering og kultur af metanfriske rudimenter fra mus.
Under udvikling af pattedyrs nyre kommunikerer de to progenitorvæv, ureterbindet og det metanephriske mesenchym, og frembringer gensidigt cellulære mekanismer til sidst at danne samlingssystemet og nephronerne i nyrerne. Da pattedyrsfostre vokser intrauterin og derfor er utilgængelige for observatøren, er der udviklet en orgelkultur. Med denne metode er det muligt at studere epithelial-mesenchymale interaktioner og cellulær adfærd under nyreorganogenese. Desuden kan oprindelsen af medfødte nyrer og urogenitale malformationer undersøges. Efter omhyggelig dissektion overføres de metanephriske rudimenter til et filter, der flyder på dyrkningsmedium og kan opbevares i en cellekultur-inkubator i flere dage. Men man må være opmærksom på, at betingelserne erKunstig og kunne påvirke stofskiftet i vævet. Også penetrering af teststoffer kunne begrænses på grund af den ekstracellulære matrix og basalmembran til stede i eksplantatet.
En væsentlig fordel ved organkultur er, at eksperimentøren kan få direkte adgang til orgelet. Denne teknologi er billig, enkel og giver mulighed for et stort antal ændringer, såsom tilsætning af biologisk aktive stoffer, undersøgelse af genetiske varianter og anvendelse af avancerede billeddannelsesteknikker.
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
Dette manuskript beskriver en metode til at isolere de udviklende metanephriske anlagen fra museembryoen og til at dyrke organets rudimenter. Denne metode er en standardteknik, som udviklet af Grobstein 8 og Saxén 9 , 10 , og blev tilpasset og modificeret af mange andre 11 , 12 . Fremgangsmetoden afhænger hovedsageligt af dissektionens varighed, idet eksplanterende overlevelse o…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne takker Leif Oxburgh og Derek Adams for generøst at dele deres viden, Leif Oxburgh for de nyttige bemærkninger til manuskriptet, og Stefan Wölfl og Ulrike Müller for deres tekniske support og Saskia Schmitteckert, Julia Gobbert, Sascha Weyer og Viola Mayer for hjælp i lab. Dette arbejde blev støttet af Development, The Company of Biologists (til CP).
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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