Sebinger Culture: A System Optimized for Morphological Maturation and Imaging of Cultured Mouse Metanephric Primordia

Abstract

Here, we present a detailed protocol on setting up embryonic renal organ cultures using a culture method that we have optimised for anatomical maturation and imaging. Our culture method places kidney rudiments on glass in a thin film of medium, which results in very flat cultures with all tubules in the same image plane. For reasons not yet understood, this technique results in improved renal maturation compared to traditional techniques. Typically, this protocol will result in an organ formed with distinct cortical and medullary regions as well as elongated, correctly positioned loops of Henle. This article describes our method and provides detailed advice. We have published qualitative and quantitative evaluations on the performance of the technique in Sebinger et al. (2010) and Chang and Davies (2012).

Keywords: Imaging; Kidney; Metanephros; Organ culture; Organoid; Sebinger culture.

Figure 1.. Kidneys cultured in the Sebinger system.

A. Bright field images for E11.5 kidney grown in culture for 0, 3 and 7 days. Scale bars = 0.5 mm. B-D. E11.5 kidneys cultured for 7 days in the Sebinger system and stained for different renal markers to show maturation. B. Stained for the ureteric bud marker CALB (shown in green) and the basement membrane marker Laminin (shown in red). The red channel shows the presence of loops of Henle dipping into the medulla; C. Stained for CALB (green) and the proximal tubular marker LTL (red); D. Stained for ECAD (ureteric bud and distal tubular marker; shown in green) and WT1 (podocyte and cap mesenchyme marker; shown in red). Scale bars = 100 μm.

Figure 2.. Illustration of the E11.5 metanephric kidney isolation.

A. The pregnant uterus; B. Squeezing embryos out of the uterus with forceps; C. The extracted E11.5 embryos; D. The caudal part of the embryo; E. The rear of the embryo, after cutting it sagittally into two halves; the arrow points towards the metanephric kidney, edges of which are marked in white in the detail (seeing the kidney is the hardest part of the dissection, and takes practise). F. The isolated E11.5 metanephric kidney. Scale bar for A-C is 5 mm, for D and E is 1 mm, and 0.5 mm for F.

Figure 3.. The Sebinger culture method.

A. Shows the different component of the culture system. B. Shows the assembled Sebinger culture system.

Figure 4.. The arrangement of the cone and dishes.

A. Kidney rudiments are cultured in the centre of the silicone cone in a low volume of KCM medium and a humidifying buffer is added to the outer dish to prevent dryness. B. The KCM tends to bead as a single drop (left hand side image) and need to be distributed with a pipette tip to cover the glass circle enclosed by the silicone cone (right hand side image).