Name: Dissection and Culture of Mouse Embryonic Kidney (Video) | JoVE Journal
Uploaded: 2017-05-17T15:00:00.000+00:00
Duration: 8 min 30 s
Description: Learn to isolate and culture metanephric rudiments from mouse embryos for studying ureteric bud branching morphology. This simple, inexpensive method enables direct observation of kidney organogenesis and congenital malformation mechanisms.

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5 minutes

The authors thank Leif Oxburgh and Derek Adams for generously sharing their knowledge, Leif Oxburgh for the helpful comments on the manuscript, and Stefan W&#246;lfl and Ulrike M&#252;ller for their technical support and Saskia Schmitteckert , Julia Gobbert, Sascha Weyer and Viola Mayer for help in the lab. This work was supported by Development, The Company of Biologists(to CP).

Mice were maintained according to Swedish regulations and European Union legislation (2010/63/EU). All procedures were performed following the guidelines of the Swedish Ethics Committee (permits C79/9, C248/11, and C135/14). Procedures at Heidelberg University involving animal subjects have been approved by the Regierungspr&#228;sidium Karlsruhe and the Animal Welfare Officers at the University of Heidelberg.
1. Preparation of Reagents and Materials for Culture
NOTE : Use a laminar flow hood to minimize contamination.
<ol>
	<li>On the day of dissection, pre-cluster human Fc alone or recombinant chimeric ephrin protein fused to human Fc by mixing it with anti-human Fc goat antibodies at a molar ratio of 1:5 in sterile phosphate-buffered saline (PBS). Incubate for 1 h at 37 &#176;C5.</li>
	<li>Prepare the culture medium by supplementing Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) with 1% penicillin/streptomycin (v/v), 1% glutamine (v/v), 1% insulin-transferrin-selenium (v/v), 50 &#181;g/mL human holo-transferrin, and 1.5 &#181;g/mL amphotericin. 
	NOTE: 10 mL of medium is sufficient for 16 embryos.</li>
	<li>Add clustered recombinant ephrin solution at a final concentration of 20 &#181;g/mL to the culture medium. Use clustered human Fc as control.</li>
	<li>Prepare culture plates by adding 500 &#181;L of culture medium to each well of a sterile, uncoated, flat-bottom polystyrene 4-well plate.</li>
	<li>Using sterile forceps, carefully place one polycarbonate membrane filter with a pore size of 5 &#181;m per well on top of the medium. Transfer the prepared plate to the incubator (37 &#176;C/5% CO2). Ensure that the filter stays dry on the upper surface and floats. 
	NOTE: One filter is sufficient for two kidney anlagen.</li>
	<li>Using a razor blade, cut approximately 30 pipette tips (volume: 100 &#181;L) approximately 1 mm from the tip to result in a wider opening. Place the tips back into a pipette-tip rack.</li>
</ol>
2. Dissection of Metanephric Rudiments at E11.5
<ol>
	<li>Sacrifice a timed-pregnant mouse at E11.5 by cervical dislocation (or using a method approved by the local ethics committee).</li>
	<li>Remove the uterus and the embryos, as described by Brown et al 6. From this point forward, work under a dissection microscope.</li>
	<li>Using No. 5 watchmaker forceps, cut the embryo directly under the forelegs. Use a fresh petri dishfor each embryo. Remove the legs and the tail of the embryo. To do this, grab the tissue to be removed with one pair of No. 5 watchmaker forceps and use the tips of a second pair to cut.</li>
	<li>To remove the ventral organ mass, use the tips of a pair of forceps to laterally open the body wall of the embryo in a rostral-to-caudal direction.
	<ol>
		<li>Cut superficially, parallel to the dorsal aorta. Use the blood-filled and therefore visible dorsal aorta for orientation. Carefully separate the ventral part of the body wall from the dorsal part using the tips of closed forceps and flip the embryo over. Cut the body wall laterally in a rostral-to-caudal direction and using the dorsal aorta for orientation, as for the previous side.</li>
	</ol>
	</li>
	<li>Once the ventral body wall has been separated from the dorsal body, grab it with one pair of forceps and carefully pull to remove it, together with the organ mass. 
	NOTE: The metanephric anlagen usually stay attached to the dorsal body wall. They are oval, ~ 200 &#181;m long structures located at the level of the hind limbs.</li>
	<li>Hold the dorsal body wall with one pair of forceps, with the ventral side facing up. Using the other pair of forceps, grab the dorsal aorta at its rostral end and carefully peel the dorsal aorta from the dorsal body wall. 
	NOTE: Both metanephric kidneys usually stay attached to the dorsal aorta.</li>
	<li>Using one pair of forceps, cut rostrally to the kidney anlagen to remove the aorta. Then, carefully separate the two kidney anlagen using the tips of the forceps.</li>
	<li>Carefully peel off unwanted tissue from the kidney anlagen using the tips of the forceps. Take care not to damage the mesenchyme, as damage can result in poor growth and exclusion of the explant from further analysis.</li>
	<li>Transfer the kidney anlagen separately using a microliter pipette and a wide-open pipette tip in 10 &#181;L of PBS to the polycarbonate membrane filter in a 4-well-plate. 
	NOTE: Due to surface tension, the explant will be covered with a thin layer of medium. One filter is sufficient for two kidney anlagen.
	<ol>
		<li>Place each of the two kidney anlagen at the center of each respective half of the filter. Transfer the plate back to the incubator at 37 &#176;C/5% CO2. Leave the plate in the incubator until the metanephric rudiments from the next embryo are ready to be placed on the filter.</li>
	</ol>
	</li>
	<li>Repeat steps 2.3-2.9.1 for each embryo. 
	NOTE: With some practice, the dissection procedure will take about 5 min per embryo.</li>
	<li>Incubate the kidney anlagen for 3 days at 37 &#176;C/5% CO2.</li>
</ol>
3. Preparation of Reagents for Fixation and Staining
<ol>
	<li>To prepare 4% paraformaldehyde (PFA) in PBS without calcium and magnesium (w/v), dissolve the PFA at 60 &#176;C in PBS, continuously stirring. Cool to room temperature and use a paper filter to remove the remaining particles. Aliquot and keep at 4 &#176;C for immediate use or freeze for long-term storage. 
	CAUTION: PFA is toxic. Wear the personal protective equipment specified in the local safety standards and work in a fume hood. Discard waste following institutional guidelines and using designated waste containers.</li>
	<li>To prepare permeabilization solution, dilute Triton X-100 in PBS without calcium and magnesium to 0.3% (v/v).</li>
	<li>To prepare blocking solution, add 5% (v/v) goat serum and 0.1% Triton X-100 to PBS without calcium and magnesium. Add sodium azide to a final concentration of 0.02% (w/v). Store at 4 &#176;C. 
	CAUTION: Sodium azide is toxic. Handle sodium azide in accordance with the local safety and environmental protection regulations. 
	NOTE: When using antibodies for staining, use 5% serum from the species the secondary antibody was derived from to make blocking solution.</li>
	<li>Dilute biotinylated Dolichorus biflorus agglutinin 1:200 in blocking solution. Dilute Alexa488-conjugated streptavidin 1:200 in blocking solution.</li>
	<li>To make ready-to-use embedding medium (see the table of materials), add 0.1 g of water-soluble polyvinyl alcohol-based mucoadhesive/mL to 25% (w/v) glycerol in 0.1 M Tris-HCl (pH 8.5). Heat to 50 &#176;C under continuous agitation for 1 h, cool down, and adjust the pH to 8.0-8.5 using 1 M NaOH. Avoid higher NaOH concentrations. Add 100 &#181;g/mL N-propylgallate and thimerosal to a final concentration of 0.02% (w/v). Stir for 30 min at room temperature. 
	CAUTION: Thimerosal is toxic. Handle it in accordance with the local safety and environmental protection regulations.
	<ol>
		<li>Fill the embedding medium in 50 mL tubes, balance the rotor, and centrifuge at 3,200 x g and room temperature for 10 min. Decant the clear solution into 15 mL tubes and discard the pellet. Aliquot and store at -20 &#176;C for several weeks. Once thawed, keep the solution at 4 &#176;C. Warm to ~30 &#176;C immediately before use.</li>
	</ol>
	</li>
	<li>Prepare as many glass slides as filters with explants are to be mounted. For this, glue two small coverslips, 18 x 18 mm, on either side of the glass slide using instant adhesive, Leave a ~15 mm space for the filter in between.</li>
</ol>
4. Fixation and staining
<ol>
	<li>After a culturing period of 3 days in vitro (div), remove the plates from the incubator and carefully aspirate the culture medium from each well using a micropipette. Make sure not to touch the filter and keep the tip opening towards the wall of the well.</li>
	<li>Add 500 &#181;L of 4% PFA/PBS to each well. The filters will float on the fixative. Using a micropipette, carefully add the PFA fixative dropwise to submerge the filters. Incubate at room temperature for 30 min. 
	NOTE: For all following steps, care must be taken not to wash off the explants from the filter. Keep the opening of the pipette tip towards the wall of the well.</li>
	<li>Remove the PFA and rinse twice with 500 &#181;L of PBS, submerging the filter. Add 500 &#181;L of permeabilization solution to each well and incubate at room temperature for 1 h.</li>
	<li>Rinse twice with 500 &#181;L of PBS and add 500 &#181;L of blocking solution to each well. Incubate at room temperature for 1 h.</li>
	<li>Replace the blocking solution with 250 &#181;L of biotinylated Dolichorus biflorus agglutinin diluted 1:200 in blocking solution and incubate at 4 &#176;C for 24 h.</li>
	<li>Remove the Dolichorus biflorus agglutinin solution and rinse twice with 500 &#181;L of PBS per well.</li>
	<li>Add 250 &#181;L of Alexa488-conjugated streptavidin diluted 1:200 in blocking solution and incubate at 4 &#176;C for 24 h. Alternatively, incubate at room temperature for 2 h. 
	NOTE: A better signal-to-noise ratio is achieved when incubated at 4 &#176;C.</li>
	<li>Rinse twice with 500 &#181;L of PBS per well. Using forceps, transfer the filters to the prepared glass slides, with the explants facing up. Mount with ~50-100 &#181;L of embedding medium. Cover with 60 mm-long No. 1.5 coverslips. Allow the embedding medium solution to solidify for 2 h at room temperature in the dark. Proceed to imaging or store the slides, wrapped in foil, at 4 &#176;C until ready to image.</li>
	<li>Image with a widefield epifluorescence microscope7 coupled to an Hg-lamp and use a mirror unit for blue excitation (excitation bandpass, 460 - 495 nm; dichromatic mirror, 505 nm; and emission bandpass, 510 - 550 nm) and 20X Plan-Apochromat lenses, 0.75 numerical aperture.</li>
</ol>

<ol><li>Saxén, L. <a target="_blank" href="http://admin.cambridge.org/fk/academic/subjects/life-sciences/cell-biology-and-developmental-biology/organogenesis-kidney">Organogenesis of the kidney. Developmental and Cell Biology Series</a>. 19, Cambridge University Press. (1987).</li>
<li>Lindström, N. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrated+%CE%B2-catenin%2C+BMP%2C+PTEN%2C+and+Notch+signalling+patterns+the+nephron%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Integrated β-catenin, BMP, PTEN, and Notch signalling patterns the nephron.</a> eLife. 4, e04000(2015).</li>
<li>Peuckert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multimodal+Eph%2FEphrin+signaling+controls+several+phases+of+urogenital+development%5BTitle%5D)+AND+%22Kidney+Int%22%5BJournal%5D)">Multimodal Eph/Ephrin signaling controls several phases of urogenital development.</a> Kidney Int. 90 (2), 373-388 (2016).</li>
<li>Pasquale, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eph+receptor+signalling+casts+a+wide+net+on+cell+behaviour%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Eph receptor signalling casts a wide net on cell behaviour.</a> Nat Rev Mol Cell Biol. 6 (6), 462-475 (2005).</li>
<li>Bonanomi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ret+Is+a+Multifunctional+Coreceptor+that+Integrates+Diffusible-+and+Contact-Axon+Guidance+Signals%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Ret Is a Multifunctional Coreceptor that Integrates Diffusible- and Contact-Axon Guidance Signals.</a> Cell. 148 (2), 568-582 (2012).</li>
<li>Brown, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+Culture+of+Cells+from+the+Nephrogenic+Zone+of+the+Embryonic+Mouse+Kidney%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney.</a> J Vis Exp. (50), e2555(2011).</li>
<li>Olympus Support. , Available from:
 <a target="_blank" href="http://www.olympusamerica.com/cpg_section/cpg_archived_product_details.asp?id=817">http://www.olympusamerica.com/cpg_section/cpg_archived_product_details.asp?id=817</a> (2016).</li>
<li>Grobstein, C. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/jez.1401300207/abstract">Inductive interaction in the development of the mouse metanephros.</a> J Exp Zool. 130, 319-340 (1955).</li>
<li>Saxén, L., Toivonen, S. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/nlmcatalog/38101">Primary Embryonic Induction</a>. , Academic Press. London. (1962).</li>
<li>Saxén, L., Koskimies, O., Lahti, A., Miettinen, H., Rapola, J., Wartiovaara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+of+kidney+mesenchyme+in+an+experimental+model+system%5BTitle%5D)+AND+%22Adv+Morphog%22%5BJournal%5D)">Differentiation of kidney mesenchyme in an experimental model system.</a> Adv Morphog. 7, 251-293 (1968).</li>
<li>Dudley, A. T., Godin, R. E., Robertson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+between+FGF+and+BMP+signaling+pathways+regulates+development+of+metanephric+mesenchyme%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">Interaction between FGF and BMP signaling pathways regulates development of metanephric mesenchyme.</a> Genes Dev. 13, 1601-1613 (1999).</li>
<li>Perälä, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sema4C-Plexin+B2+signalling+modulates+ureteric+branching+in+developing+kidney%5BTitle%5D)+AND+%22Differentiation%22%5BJournal%5D)">Sema4C-Plexin B2 signalling modulates ureteric branching in developing kidney.</a> Differentiation. 81 (2), 81-91 (2011).</li>
<li>Thesleff, I., Ekblom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+transferrin+in+branching+morphogenesis%2C+growth+and+differentiation+of+the+embryonic+kidney%5BTitle%5D)+AND+%22J+Embryol+exp+Morph%22%5BJournal%5D)">Role of transferrin in branching morphogenesis, growth and differentiation of the embryonic kidney.</a> J Embryol exp Morph. 82, 147-161 (1984).</li>
<li>Watanabe, T., Costantini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+analysis+of+ureteric+bud+branching+morphogenesis%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Real-time analysis of ureteric bud branching morphogenesis.</a> Dev Biol. 271, 98-108 (2004).</li>
<li>Sebinger, D. D. R., Unbekandt, M., Ganeva, V. V., Ofenbauer, A., Werner, C., Davies, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Novel%2C+Low-Volume+Method+for+Organ+Culture+of+Embryonic+Kidneys+That+Allows+Development+of+Cortico-Medullary+Anatomical+Organization%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A Novel, Low-Volume Method for Organ Culture of Embryonic Kidneys That Allows Development of Cortico-Medullary Anatomical Organization.</a> PLoS One. 5 (5), e10550(2010).</li>
<li>Ekblom, P., Miettinen, A., Virtanen, I., Wahlström, T., Dawnay, A., Saxén, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+segregation+of+the+metanephric+nephron%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">In vitro segregation of the metanephric nephron.</a> Dev Biol. 84 (1), 88-95 (1981).</li>
<li>Davies, J. A., Unbekandt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((siRNA-mediated+RNA+interference+in+embryonic+kidney+organ+culture%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">siRNA-mediated RNA interference in embryonic kidney organ culture.</a> Methods Mol Biol. 886, 295-303 (2012).</li>
<li>Saxén, L., Lehtonen, E. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/3328727">Embryonic kidney in organ culture.</a> Differentiation. 36 (1), 2-11 (1987).</li>
<li>Bard, J. B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+development+of+the+mouse+kidney+embryogenesis+writ+small%5BTitle%5D)+AND+%22Curr+Opin+Genet+Dev%22%5BJournal%5D)">The development of the mouse kidney embryogenesis writ small.</a> Curr Opin Genet Dev. 2, 589-595 (1992).</li>
<li>Davies, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+for+cold+storage+and+transport+of+viable+embryonic+kidney+rudiments%5BTitle%5D)+AND+%22Kidney+Int%22%5BJournal%5D)">A method for cold storage and transport of viable embryonic kidney rudiments.</a> Kidney Int. 70 (11), 2031-2034 (2006).</li>
<li>Batourina, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distal+ureter+morphogenesis+depends+on+epithelial+cell+remodeling+mediated+by+vitamin+A+and+Ret%5BTitle%5D)+AND+%22Nat+Genet%22%5BJournal%5D)">Distal ureter morphogenesis depends on epithelial cell remodeling mediated by vitamin A and Ret.</a> Nat Genet. 32 (1), 109-115 (2002).</li>
</ol>

The authors have nothing to disclose.

This manuscript describes a method to isolate the developing metanephric anlagen from the mouse embryo and to culture the organ rudiments. This method is a standard technique, as developed by Grobstein8 and Sax&#233;n9,10, and was adapted and modified by many others11,12. The success of the method depends mainly on the duration of the dissection, as explant survival and inductive p...

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 Sax&#233;n, 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 Sax&#233;n'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.

<table><tbody><tr><td>DMEM/F-12</td><td>Thermo Fisher Scientific</td><td>21331020</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo Fisher Scientific</td><td>15140148</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Thermo Fisher Scientific</td><td>35050061</td><td></td></tr><tr><td>DPBS, calcium, magnesium</td><td>Thermo Fisher Scientific</td><td>14040117</td><td>use for dissection</td></tr><tr><td>holo-Transferrin human</td><td>Sigma-Aldrich</td><td>T0665</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium (ITS -G) (100x)</td><td>Thermo Fisher Scientific</td><td>41400045</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Amphotericin B solution</td><td>Sigma-Aldrich</td><td>A2942</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S8032</td><td></td></tr><tr><td>Thimerosal</td><td>Sigma-Aldrich</td><td>T5125</td><td></td></tr><tr><td>Propyl gallate</td><td>Sigma-Aldrich</td><td>2370</td><td></td></tr><tr><td>Mowiol 4-88</td><td>Sigma-Aldrich</td><td>81381</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Biotinylated Dolichorus Biflorus Agglutinin</td><td>Vector Laboratories</td><td>B-1035</td><td></td></tr><tr><td>Alexa488 conjugated Streptavidin</td><td>Jackson Immuno Research</td><td>016-540-084</td><td></td></tr><tr><td>Recombinant Mouse Ephrin-B2 Fc Chimera Protein, CF</td><td>R&amp;amp;D Systems</td><td>496-EB</td><td></td></tr><tr><td>Recombinant Human IgG1 Fc, CF</td><td>R&amp;amp;D Systems</td><td>110-HG-100</td><td></td></tr><tr><td>Goat Anti-Human IgG Fc Antibody</td><td>R&amp;amp;D Systems</td><td>G-102-C</td><td></td></tr><tr><td>Phosphate buffered saline tablets</td><td>Sigma-Aldrich</td><td>P4417</td><td>use for fixation and immunostaining</td></tr><tr><td>Dumont #5, biologie&lt;br/&gt; tips, INOX, 11 cm</td><td>agnthos.se</td><td>0208-5-PS</td><td>2 pairs of forceps are needed</td></tr><tr><td>Iris scissors, straight, 12 cm</td><td>agnthos.se</td><td>03-320-120</td><td></td></tr><tr><td>Dressing Forceps,&lt;br/&gt; straight, delicate, 13 cm</td><td>agnthos.se</td><td>08-032-130</td><td></td></tr><tr><td>Petri dishes Nunclo Delta treated</td><td>Thermo Fisher Scientific</td><td>150679</td><td></td></tr><tr><td>TMTP01300 Isopore Membrane Filter, polycarbonate, Hydrophilic, 5.0 &amp;micro;m, 13 mm, white, plain</td><td>MerckMillipore</td><td>TMTP01300</td><td></td></tr><tr><td>Nunclon Multidishes&lt;br/&gt; 4 wells, flat bottom</td><td>Sigma-Aldrich</td><td>D6789-1CS</td><td></td></tr><tr><td>Microscope cover glass 24 x 50 mm thickn. No.1.5H 0.17+/-0.005 mm</td><td>nordicbiolabs</td><td>107222</td><td></td></tr><tr><td>Cover glasses No.1.5, 18 mm x 18 mm</td><td>nordicbiolabs</td><td>102032</td><td></td></tr><tr><td>Slides ~76 x 26 x 1, 1/2-w. ground plain</td><td>nordicbiolabs</td><td>1030418</td><td></td></tr><tr><td>VWR Razor Blades</td><td>VWR</td><td>55411-055</td><td></td></tr><tr><td>50 mL centrifuge tubes</td><td>Sigma-Aldrich</td><td>CLS430828</td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td>Sigma-Aldrich</td><td>CLS430055</td><td></td></tr><tr><td>Whatman prepleated qualitative filter paper, Grade 113V, creped</td><td>Sigma-Aldrich</td><td>WHA1213125</td><td></td></tr><tr><td>Fixed stage research mircoscope</td><td>Olympus</td><td>BX61WI</td><td></td></tr><tr><td>Black 6 inbred mice, male, C57BL/6NTac</td><td>Taconic</td><td>B6-M</td><td></td></tr><tr><td>Black 6 inbred mice,female, C57BL/6NTac</td><td>Taconic</td><td>B6-F</td><td></td></tr><tr><td>Greenough Stereo Microscope</td><td>Leica</td><td>Leica S6 E</td><td></td></tr></tbody></table>

dissection and culture of mouse embryonic kidney

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

Metanephric kidney anlagen were derived from pregnant Black-6 inbred mice at E11.5 and were cultured. After 3 days, the ureteric bud had branched up to 5 times, resulting in a ramification of the initially T-shaped ureteric bud. Each explant was photographed, and the numbers of segments and endpoints were quantified to determine the branching generations and to calculate the number of endpoints per branch (Figure 1). ImageJ (rd generation; the 4th ge...

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

The overall goals of this procedure are to isolate and culture metanephric rudiments from mouse embryos and to observe the effects of specific treatments on ureteric bud branching morphology. This method can help answer key questions in kidney organogenesis about the control of tubular branching morphogenesis, about epithelial mesenchyme interactions, and about the origin of congenital malformations. The main advantages with this technique are that it facilitates direct access to the organ;that it is simple, inexpensive, and easily modified.Before beginning the dissection, use sterile forceps to carefully place one polycarbonate membrane filter with a five-micrometer pore size into each well of a four-well plate containing 500 microliters of fresh culture medium. Transfer the plate into a cell culture incubator. Then use a razor blade to cut approximately 30 100-microliter micropipette tips approximately one millimeter from the tip to widen the tip openings and return the tips to the pipette tip rack.When the tips are ready, place an embryonic day 11 1/2 embryo in PBS under a dissecting microscope. Use a fresh dish for each embryo. And with number five watchmaker forceps, cut the embryo directly under the forelegs.Next, hold the legs with one pair of forceps using a second forceps to remove the leg tissue. Remove the tail in the same manner, and use the tips of the forceps to laterally and superficially open the body wall of the embryo in a rostral and caudal direction parallel to the dorsal aorta. Cutting superficially, make an incision along the dorsal aorta and use the tips of the closed forceps to carefully separate the ventral part of the body wall from the dorsal body wall.Flip the embryo over and cut the body wall laterally in a rostral-to-caudal direction using the dorsal aorta for orientation as just demonstrated. Once the ventral body wall has been separated from the dorsal body, grasp the ventral body wall with the forceps, and carefully pull to remove the wall and the ventral organ mass. Holding the dorsal body wall with one forceps with the ventral side facing up, use the other forceps to grasp the dorsal aorta at its rostral end and carefully peel the dorsal aorta from the dorsal body wall.Using one forceps, cut rostrally to the kidney anlagen to remove the aorta. And use the tips of the forceps to carefully separate the two anlagen. Use the tips of the forceps to carefully peel the unwanted tissue from the kidney anlagen.And use a microliter pipette and a modified pipette tip to transfer one anlagen and 10 microliters of PBS into the center of one half of a single polycarbonate membrane filter in the four-well plate. Then return the plate to the incubator until the metanephric rudiments from the next embryo are ready to be placed on the filter. After three days at 37 degrees Celsius and 5%carbon dioxide, use a micropipette to carefully replace the culture medium in each well with 500 microliters of 4%paraformaldehyde and PBS dropwise to submerge the filters.After 30 minutes at room temperature, remove the fixative and rinse the filters two times with 500 microliters of PBS submerging the filters with each wash. Then add 500 microliters of permeabilization solution to each well for a one-hour incubation at room temperature. At the end of the permeabilization incubation, rinse the filters two times as just demonstrated, and add 500 microliters of blocking solution to each well, After one hour at room temperature, replace the blocking solution with 250 microliters of biotinylated Dolichos biflorus agglutinin and blocking solution, and place the tissues at four degrees Celsius for 24 hours.The next day, wash the filters two times with 500 microliters of PBS per well. And label the tissue with 250 microliters of Alexa 488 conjugated streptavidid and blocking solution for 24 hours at four-degree Celsius. The next day, wash the tissues two times with 500 microliters of PBS per well and use forceps to transfer the filters to prepared glass slides with the explants facing up.Mount the explants with 50 to 100 microliters of embedding medium and cover the tissues with 60-millimeter long number 1.5 coverslips. Then allow the embedding medium to solidify for two hours at room temperature in the dark, and image the metanephric kidney samples. After three days of culture, the ureteric bud of the metanephric kidney anlagen branches up to five times, resulting in a ramification of the initially T-shaped ureteric bud.In the presence of clustered ephrin-B2, the branching complexity has decreased, as compared to control explants incubated with clustered human Fc.Indeed, ureteric bud branching in the majority of the ephrin-B2 treated explants, does not exceed third generation. And the fourth generation is reached in only 8%of the treated explants. Accordingly, the number of endpoints per branch and endpoints per millimeter squared is reduced in the explants treated with clustered ephrin-B2.Further, a third of the explants demonstrate an unusual morphology in the ureteric bud tips, suggesting that ephrin-B2 might have a restricting effect on ureteric bud branching process, most likely by activating FA4 and FB2 forward signaling. Any injury of the mesenchyme decreases the inductive potential leading to a reduced or absent ureteric bud branching. For example, in this explant with an almost-missing mesenchyme, the ureteric bud did not branch beyond the T stage.In this experiment, the metanephric mesenchyme was damaged, leading to poor growth and branching. After it's development in the 1960s, this technique paved the way for researchers in the field of developmental biology to explore kidney organogenesis. After watching this video, you should have a good understanding of how to isolate and culture metanephric rudiments from E11.5 mouse embryo.Once mastered, the dissection can be completed in less than five minutes if performed properly.

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Research

JoVE Journal

Developmental Biology

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Learn to isolate and culture metanephric rudiments from mouse embryos for studying ureteric bud branching morphology. This simple, inexpensive method enables direct observation of kidney organogenesis and congenital malformation mechanisms.

Dissection and Culture of Mouse Embryonic Kidney

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