Rodent Kidney Explant Culture and Establishment of Primary Ureteric Bud Epithelial Cell Culture

Summary

This protocol describes the establishment of the most commonly used mammalian kidney culture, which allows renal growth and differentiation in a Trowell-type air-liquid interphase system. We also detail how to set up primary ureteric bud epithelial cell cultures from the early kidney rudiments.

Abstract

The developing kidney is a widely used model system to study mammalian organogenesis. Renal differentiation is orchestrated by reciprocal inductive tissue interactions among mesenchyme, stroma, and epithelium. Branching morphogenesis of epithelial ureteric bud promotes not only embryonic kidney growth and 3D patterning but also induces mesenchyme-to-epithelium transitions within the surrounding nephron progenitor pool, thereby ensuring nephron formation required for functional filtration.

Critical cell fate decisions within all three tissue layers, epithelium, mesenchyme, and stroma, underpin a dynamic balance between progenitor self-renewal and differentiation equilibrium. These complex, multilayered regulatory interactions are inherently dependent on the structural integrity of the organ, making intact kidney models essential for studying morphogenetic processes and cellular behaviors in their native context.

Kidney organ culture methods provide insightful platforms for visualizing developmental processes and dissecting morphological, cellular, and molecular mechanisms that govern renal differentiation. A variety of kidney culture methods exist, each offering distinct advantages. In this article, we detail the establishment of a traditional Trowell-type air-liquid interphase culture system for in vitro kidney development. To enhance the resolution of cellular and molecular analyses, we also describe the setup of a primary ureteric bud epithelial cell culture, which enables targeted investigation of epithelial-specific mechanisms.

Finally, we demonstrate that the traditional kidney organ culture technique is applicable to the embryonic kidney of a novel model organism, the spiny mouse (Acomys dimidiatus). This opens interesting innovative avenues for stem cell and regeneration research, as Acomys has a remarkable functional repair capacity across several organ systems studied to date.

Introduction

The practice of culturing organs dates back to the beginning of the 20^th century¹. Kidneys were among the first organs to be cultured in vitro when Rienhoff introduced the hanging drop method for embryonic chicken kidneys in 1922². In 1950's, embryonic mouse (Mus musculus) kidney cultures established the importance of mesenchyme-epithelium interactions in guiding developmental processes^3,4. Since then, tissue and organ cultures have served as a method to study branching morphogenesis^5,

Protocol

All experiments were performed in accordance with relevant national and European Union guidelines and regulations. The Committee for animal experiments of the University of Helsinki approved the use of rodents for these experiments under the licenses KEK22-016 (valid: 1.7.2022-30.6.2025) and KEK25-014 (valid: 1.7.2025-30.6.2028).

NOTE: Make sure to dispose of all sharps and chemicals according to institutional guidelines and good laboratory practices. Kidney cultures can be established with early kidney rudiments originating from rodent embryos. The typical stages for setting up the kidney culture are E11.5-E13.5. The earlier the organ is, ....

Results

The ex vivo kidney culture method can be used to address a wide array of research questions related to kidney development, and the outcome of each experiment depends on the experimental setup. However, with the use of appropriate control samples, it is important to ensure that the culture conditions themself do not affect the development. Ideally, the control samples ex vivo recapitulate the in vivo kidney development. The house mouse has been the standard model of kidney culture experiments. W.......

Discussion

The kidney is a vital organ with significant blood filtration and body fluid balancing functions. The basis for renal functional capacity is built during fetal life, when all nephrons for life are formed^44,51,97. Understanding of both the kidney growth through branching morphogenesis and the nephron differentiation per se have extensively benefited from kidney culture methods, which have provided the fundamental insight.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10X PBS	Lonza Biosciences	51226	1X solution for the use
1ml syringes	Thermo Fisher Scientific	10564431
23-25G disposable injection needles	Thermo Fisher Scientific	10482792
24-well plates	Greiner	662160
6-well CellStar plates	Merck	M8562
70% ethanol (EtOH), diluted in MilliQ-H2O	Any commericial provider		Used for sterilizing surfaces and skin
Bovine serum albumin (BSA)	Thermo Fisher	B14	Final concentration of 0.2%
Collagenase	Invitrogen	17018029	200mg/ml stock in PBS
DMEM/F12	Thermo Fisher	31331028
Dnase	Fisher	10649890
Drummond Wiretrol II Micro Dispenser, 10-20µl	Transcat pipettes	DRMC064
Dulbecco’s PBS (with Ca2+ and Mg2+)	Thermo Fisher Scientific	14040117	Dulbecco’s PBS is interchangeable with CO2 independent medium (Gibco/ThermoFisher), where embryos and tissues are well-protected for short- and long-term storage.
E11.5 - E12.5 mouse embryos	Any commericial breeder / own stock	https://janvier-labs.com/en/	Mouse embryos are typically produced by in-house plugging. The day of vaginal plug appearance is E0.5. Commercial  female mice with timed pregrancies are also available.
E18-19 spiny mouse embryos	N/A	N/A	Corresponding alternative for mouse embryos.
Fetal bovine serum (FBS)	Gibco	A5256801
Fibronectin	Novus Biologicals	1030-FN-05M	1mg/ml stock
Glass or plastic dishes (100mm, 60mm and 30mm).	Gibco	150476, 174888, 153066
GlutaMAX	Gibco	35050061
human recombinant Fibroblast Growth Factor (rFGF2)	PeproTech	100-18B
Human recombinant Glial Cell Line Derived Neurotrophic Factor (rGDNF)	R&D	212-GD-010/CF
Human recombinant Hepatocyte Growth Factor (rHGF)	PeproTech	100-39
penicillin-streptomycin	Gibco	15140122
Small scissors and fine forceps	Fine Science Tools	14040-10, 11254-20	Only use clean and sterile dissection tools to avoid any source of contamination.
Stereomicroscope	Leica MZ7.5 or Olympus SZ x9	https://www.leica-microsystems.com/
Transwell Permeable Supports, 24mm inserts	Fisher	10619141