Organoid culture media formulated with growth factors of defined cellular activity

Abstract

The media formulations necessary for deriving and sustaining organoids from epithelial tissues such as prostate, colon, gastric, liver, pancreas, and others have been established. Critical components of organoid media are a set of growth factors that include R-spondins and BMP signalling antagonists such as Noggin or Gremlin 1. Currently, the practical limitations for formulating organoid media of reproducible potency and larger-scale media production that have hampered further technological applications of organoid technology include: the cost of growth factors such as R-spondins and Gremlin 1/Noggin and their production as defined specific activities free of contaminants that may affect organoid growth. Here we report the production of highly pure recombinant Gremlin 1 and R-spondin 1 from bacterial expression for use in organoid media. We detail the workflow for Gremlin 1 and R-spondin 1 expression, purification, quantification of cellular activity, quality control and use in media formulated for culturing organoids derived from a number of tissues. The development of precisely formulated, cost-effective media of defined specific activity will engender the development of novel applications for organoid technology.

Stem cell‐derived enteroid cultures as a tool for dissecting host‐parasite interactions in the small intestinal epithelium

“…The heterogeneity of the enteroid population in terms of size and differentiation may also fundamentally influence experimental results 56 . Finally, the cost of specialist growth media for maintaining enteroid cultures may be prohibitive for some research groups 57 …”

Spatiotemporal Gradient and Instability of Wnt Induce Heterogeneous Growth and Differentiation of Human Intestinal Organoids

“…The protocol of organoid culture has been optimized and widely implemented by various research groups ( Ettayebi et al., 2016 ; Li et al., 2014 ). However, due to the complex culture condition ( Holloway et al., 2019 ), inter-heterogeneity of the biopsy-derived organoids has often been observed between organoid lines ( Michels et al., 2019 ; Urbischek et al., 2019 ) or the quality of conditioned medium for supplying the major morphogens ( Fatehullah et al., 2016 ). Various efforts have been made to overcome these challenges by increasing the number of experimental replicates, testing multiple lines, and validating the effect of different passage numbers to improve experimental statistics.…”

Increased Expression of Gremlin1 Promotes Proliferation and Epithelial Mesenchymal Transition in Gastric Cancer Cells and Correlates With Poor Prognosis of Patients With Gastric Cancer

“…The positive impact on the proliferation and tumourigenic capacity of GC cells is in line with its implication in disease progression. This is also supported by a very recently published study in which showed the tumourigenic potential of Gremlin1using an organoid model (16). BMPs have been shown to inhibit proliferation of epithelial cells and epithelium derived cancerous cells through canonical signaling pathways, upon binding to their receptors (17).…”