Abstract
ABSTRACTRecent years have seen significant developments in the ability to continuously propagate organoids derived from intestinal crypts. These advancements have been applied to mouse and human samples providing models for gastrointestinal tissue development and disease. We adapt these methods for the propagation of intestinal organoids (enteroids) from various large farm and small companion (LF/SC) animals, including cat, dog, cow, horse, pig, sheep and chicken. We show that LF/SC enteroids propagate and expand in L-WRN conditioned media containing signaling factors Wnt3a, R-spondin-3, and Noggin (WRN). Multiple successful isolations were achieved for each species, and the growth of LF/SC enteroids was maintained to high passage number. LF/SC enteroids expressed crypt stem cell marker LGR5 and low levels of mesenchymal marker VIM. Labeling with EdU also showed distinct regions of cell proliferation within the enteroids marking crypt-like regions. The ability to grow and maintain LF/SC enteroid cell lines provides additional models for the study of gastrointestinal developmental biology as well as platforms for the study of host-pathogen interactions between intestinal cells and zoonotic enteric pathogens of medical importance.
Highlights
The development of primary cell lines for tissue culture has been an integral foundation to the study of cellular and molecular biology
WRN signaling molecules are conserved in large farm and small companion (LF/SC) animals Given the effectiveness of the WRN factors in stimulating the growth of both mouse and human intestinal enteroids, we were determined to test the activity of Conditioned media (CM) containing these factors on the growth of enteroids from animals of veterinary relevance
We obtained the L-WRN cell line from the American Type Culture Collection (ATCC) to produce L-WRN cell conditioned media (50% L-WRN CM), and used it to grow intestinal enteroids (Fig. 1B) (Miyoshi and Stappenbeck, 2013). This media was first tested for the growth of mouse intestinal crypt derived enteroids, which rapidly expanded after isolation from the small intestine, eventually to high passage number (P>10) (Fig. 1C), confirming the effectiveness of the media
Summary
The development of primary cell lines for tissue culture has been an integral foundation to the study of cellular and molecular biology. When cultured in a 3D matrix with media containing the WRN factors, mouse intestinal crypt cells expand into organoid structures
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