Dissection and Culture of Mouse Embryonic Kidney

Summary

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

Abstract

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.

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 me....

Protocol

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 Regierungspräsidium 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.

1. On the day of dissection, pre-cluster human Fc alone or recombinant chimeric ephrin ....

Results

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 4^th ge.......

Discussion

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 Saxén^9,10, and was adapted and modified by many others^11,12. The success of the method depends mainly on the duration of the dissection, as explant survival and inductive p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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, 11 cm	agnthos.se	0208-5-PS	2 pairs of forceps are needed
Iris scissors, straight, 12 cm	agnthos.se	03-320-120
Dressing Forceps, straight, delicate, 13 cm	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 glass 24 x 50 mm thickn. No.1.5H 0.17+/-0.005 mm	nordicbiolabs	107222
Cover glasses No.1.5, 18 mm x 18 mm	nordicbiolabs	102032
Slides ~76 x 26 x 1, 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