Triple-Decker Sandwich Cultures of Intestinal Organoids for Long-Term Live Imaging, Uniform Perturbation, and Statistical Sampling.

Three-dimensional organoid cultures enable the study of stem cell and tissue biology ex vivo, providing improved access to cells for perturbation and live imaging. Typically, organoids are grown in hydrogel domes that are simple to prepare but that lead to non-uniform tissue growth and viability. We recently developed a simple alternative culture method to embed intestinal organoids in multilayered hydrogels, called "triple-decker sandwiches," that align organoids in a common z-plane with uniform access to medium. This culture configuration improves the growth and survival of organoids over a wide working area and facilitates long-term confocal imaging and molecular perturbation. Here, we present protocols for preparing organoids in triple-decker sandwich cultures and using them for live imaging, immunostaining, and single-cell RNA sequencing. We have tested our methods on mouse and human intestinal organoids and expect them to be useful for other highly proliferative three-dimensional cell cultures.