Optimizing Wnt-3a and R-spondin1 concentrations for stem cell renewal and differentiation in intestinal organoids using a gradient-forming microdeviceElectronic supplementary information (ESI) available. See DOI: 10.1039/c5ra14923a

• Main Authors: Ahmad, Asad A, Wang, Yuli, Sims, Christopher E, Magness, Scott T, Allbritton, Nancy L
• Format: Article
• Language: English
• ISSN: 2046-2069
• DOI: 10.1039/c5ra14923a

Epithelial renewal in the colonic crypt is a tightly controlled process and essential for normal digestive function. Crypts contain both proliferative and differentiated cells with the balance orchestrated by the interplay of proliferation and differentiation signals. While critically important to homeostatic renewal, the threshold concentrations of factors such as Wnt-3a and R-spondin1 that promote stem cell renewal are unknown. A simple, linear gradient-generating device was used to screen a wide range of Wnt-3a and R-spondin1 concentrations for their impact on a large number of colon organoids (colonoids) to achieve statistically significant results while minimizing the amounts of costly reagents and primary tissue samples. Matrigel-embedded colonoids were tracked for the presence of stem/transit-amplifying cells, differentiated cells and overall colonoid size in response to varying concentrations of Wnt-3a and R-spondin1, presented singly and in combination. A Wnt-3a concentration of 60 ng mL −1 and R-spondin1 concentration of 88 ng mL −1 were identified as the critical concentrations required for stem-cell renewal and colonoid expansion. At these concentrations, the colonoids possessed similar viability, size and passage efficiency compared to colonoids cultured under standard growth concentrations. The gradient culture system enabled efficient determination of the minimal growth factor concentrations needed to produce a physiologically-relevant colonoid phenotype. Relative to traditional culture conditions, lower factor concentrations yielded the added benefit of a more morphologically appropriate colonoid possessing columnar cells surrounding a central lumen with active crypt-like bud formation. A gradient-generating device assayed the impact of Wnt-3a and R-spondin1 on colonoids identifying concentrations required to yield a physiologically-relevant epithelium.