Whole-mount Enteroid Proliferation Staining

Abstract

Small intestinal organoids, otherwise known as enteroids, have become an increasingly utilized model for intestinal biology in vitro as they recapitulate the various epithelial cells within the intestinal crypt (Mahe et al., 2013; Sato et al., 2009). Assessment of growth dynamics within these cultures is an important step to understanding how alterations in gene expression, treatment with protective and toxic agents, and genetic mutations alter properties essential for crypt growth and survival as well as the stem cell properties of the individual cells within the crypt. This protocol describes a method of visualization of proliferating cells within the crypt in three dimensions (Barrett et al., 2015). Whole-mount proliferation staining of enteroids using EdU incorporation enables the researcher to view all proliferating cells within the enteroid as opposed to obtaining growth information in thin slices as would be seen with embedding and sectioning, ensuring a true representation of proliferation from the stem cell compartment to the terminally differentiated cells of the crypt.

Figure 1. Processing of the small intestine for crypt isolation

A. After removal of the small intestine, the most proximal 10 cm should be collected for further processing. B. Then this section should be flushed with ice cold PBS using the gavage needle attached to the 10 ml syringe. C. The intestinal section is then bisected. D. The bisected intestine is then cut into approximately 1 cm sections and is transferred to a 15 ml conical tube containing 5 ml ice cold PBS.

Figure 2. Representative images of villi and crypts at 4× and 20× magnification

Red arrow heads indicate crypts. The images on the left are representative of those likely seen after step A13. The images on the right demonstrate the optimal crypt composition which will be seen after step A18.

Figure 3. Images of matrigel plating onto 12-well MatTek plates

A. Matrigel containing crypts is pipetted into the center of the well, avoiding any bubble formation. B. After the Matrigel has set, media is added to each well.

Figure 4

Representative images of enteroids 1 day, 3 days, and 5 days post-plating

Figure 5. Representative images of proliferating enteroids

Enteroids have been stained for the proliferation marker EdU (green) at (A) 3 days and (B) 5 days post-plating. Counterstain is TO-PRO-3 (nuclei, red).

Video Figure 1

Crypt isolation example