Abstract
Background & AimsDiligent side-by-side comparisons of how different methodologies affect growth efficiency and quality of intestinal colonoids have not been performed leaving a gap in our current knowledge. Here, we summarize our efforts to optimize culture conditions for improved growth and functional differentiation of mouse and human colon organoids.MethodsMouse and human colon organoids were grown in four different media. Media-dependent long-term growth was measured by quantifying surviving organoids via imaging and a cell viability readout over five passages. The impact of diverse media on differentiation was assessed by quantifying the number of epithelial cell types using markers for enterocytes, stem cells, Goblet cells, and enteroendocrine cells by qPCR and histology upon removal of growth factors.ResultsIn contrast to Wnt3a-conditioned media, media supplemented with recombinant Wnt3a alone did not support long-term survival of human or mouse colon organoids. Mechanistically, this observation can be attributed to the fact that recombinant Wnt3a did not support stem cell survival or proliferation as demonstrated by decreased LGR5 and Ki67 expression. When monitoring expression of markers for epithelial cell types, the highest level of organoid differentiation was observed after combined removal of Wnt3a, Noggin, and R-spondin from Wnta3a-conditioned media cultures.ConclusionOur study defined Wnt3a-containing conditioned media as optimal for growth and survival of human and mouse organoids. Furthermore, we established that the combined removal of Wnt3a, Noggin, and R-spondin results in optimal differentiation. This study provides a step forward in optimizing conditions for intestinal organoid growth to improve standardization and reproducibility of this model platform.
Highlights
The intestinal epithelium forms the largest of the body’s mucosal surfaces with a single layer of polarized cells covering a surface area of ~400 m2
Wnt3a-Conditioned Medium Is Essential for the Long-Term Survival of Mouse Colonoid Cultures
Mouse colonoids were grown for five passages comparing four different growth media conditions: Wnt3a-Noggin-Rspondin conditioned media (WNR CM, Figure 1A first row of images), Wnt3aconditioned media supplemented with recombinant EGF, Noggin and R-spondin (Wnt3a CM + rENR, Figure 1A second row), media with recombinant Wnt3a, EGF, Noggin, and R-spondin, or the latter medium without Wnt3a
Summary
The intestinal epithelium forms the largest of the body’s mucosal surfaces with a single layer of polarized cells covering a surface area of ~400 m2 In addition to their role in digestion and water/ nutrient resorption, intestinal epithelial cells (IECs) function as a physical and biochemical barrier to separate host tissue from commensal bacteria and digestion end products in the intestinal lumen [1]. The most common models rely on the use of colonic adenocarcinoma cell lines which retain altered cellular pathways of transformed cells Such cell cultures, in their polarized form, recapitulate some features of the intestinal epithelium and are useful for studying functions such as apical and basolateral distribution of proteins of research interest, para- and trans-cellular transport mechanisms, or the formation of tight junctions [7]. We summarize our efforts to optimize culture conditions for improved growth and functional differentiation of mouse and human colon organoids
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