BSTRACTThe GTPase Ras is a molecular switch engaged downstream of G-protein-coupled receptors and receptor tyrosine kinases that controls multiple cell-fate-determining signalling pathways.

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.

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words)The media components necessary for deriving and sustaining organoids from a number of epithelial tissues such as prostate, colon, gastric, liver, pancreas, and others have been established (1). Critical components of organoid media formulations are a set of growth factors that include EGF, R-spondins and BMP signalling antagonists such as Noggin or Gremlin. The practical limitation to organoid culture and the development of new applications for the technology is the use of defined cellular activities of growth factors in media formulations, in particular Noggin/Gremlin 1 and R-spondin 1. Here we report the production of highly pure recombinant Gremlin 1 and R-spondin 1 from bacterial expression and their use for culturing organoids. We detail the workflow for their purification, determination of cellular activity, quality control and their formulation in organoid media. The protocols we provide for generation of precisely formulated, cost-effective, organoid media of defined cellular activity will enable broader access to organoid technology and engender the development of novel applications.