Novel fixed z-direction (FiZD) kidney primordia and an organoid culture system for time-lapse confocal imaging.

Ulla Saarela,Saad Ullah Akram,Silvia Cereghini,Jingdong Shan,Janne Heikkilä,Ilya Skovorodkin,Seppo J Vainio,Veli-Pekka Ronkainen,Audrey Desgrange,Aleksandra Rak-Raszewska

doi:10.1242/dev.142950

Abstract

ABSTRACTTissue, organ and organoid cultures provide suitable models for developmental studies, but our understanding of how the organs are assembled at the single-cell level still remains unclear. We describe here a novel fixed z-direction (FiZD) culture setup that permits high-resolution confocal imaging of organoids and embryonic tissues. In a FiZD culture a permeable membrane compresses the tissues onto a glass coverslip and the spacers adjust the thickness, enabling the tissue to grow for up to 12 days. Thus, the kidney rudiment and the organoids can adjust to the limited z-directional space and yet advance the process of kidney morphogenesis, enabling long-term time-lapse and high-resolution confocal imaging. As the data quality achieved was sufficient for computer-assisted cell segmentation and analysis, the method can be used for studying morphogenesis ex vivo at the level of the single constituent cells of a complex mammalian organogenesis model system.

Highlights

High-resolution confocal imaging is an important means of studying the cellular behavioural dynamics of organ morphogenesis, a process that involves division, migration, death and changes in shape of the cells
The reasons for this are the long focal length, the thickness of the tissue, and the air-liquid interface or Transwell membrane between the specimen and the objective (Fig. 1A). This led us to consider whether restricting the ability of the sample to grow in the z-direction by use of a porous membrane (Fig. 1B) would enable better imaging quality. This proved to be optimal for supporting organogenesis and providing a high imaging quality (Fig. 2)
We found empirically that a spacer of

Summary

Introduction

INTRODUCTION High-resolution confocal imaging is an important means of studying the cellular behavioural dynamics of organ morphogenesis, a process that involves division, migration, death and changes in shape of the cells. For high-resolution time-lapse imaging, more sophisticated ways of organizing the organotypic culture experiments using an onstage microscope incubator are required. We report here a novel technique for culturing embryonic kidneys and organoids in a fixed z-direction culture (FiZD) set up in a restricted space between a Transwell filter and a glass coverslip.

Results

Conclusion