# Necessary, Legendary and Detrimental Components of Human Colorectal Organoid Culture Medium: Raising Awareness to Reduce Experimental Bugs

## TL;DR

This review highlights how components in human colorectal organoid culture medium can unintentionally affect experiments, urging researchers to be aware of their potential biases.

## Contribution

The paper identifies specific medium components that introduce experimental bias and suggests when they should be removed.

## Key findings

- Molecules like nicotinamide and PGE2 inhibit immune responses in organoid cultures.
- SB202190 and A83-01 alter intracellular signaling pathways in organoids.
- N-AcetylCysteine and Primocin affect redox and mitochondrial metabolism.

## Abstract

The invention of a culture medium that can propagate human colorectal organoids indefinitely is a milestone in the development of advanced physiological and pathological models. Although this medium is highly effective, its formula contains molecules that affect cell behavior through direct and off-target effects. Unfortunately, most researchers are unaware of the potential risks these molecules pose to their assays. This short review aims to provide a simple and effective overview of the multiple pathways and biological models that are actively modulated by the organoid medium. It also suggests which components should be eliminated under specific conditions to avoid biased results.

The creation of a specific culture medium for colorectal organoids in 2011 heralded a new era in human primary cultures by enabling the indefinite expansion of normal and pathological epithelial organoids. The original formula has been used ever since, with only minor, lab-specific modifications. The goal of culturing organoids from different tissues has relied on saving and propagating the pluripotent stem cell. The “magic bullet” and all its subsequent derivatives have pursued this goal. Consequently, agonist and antagonist signals are chronically activated in the organoid medium, forcing organoid cells (as well as any other co-cultured cellular model) into constrained signaling pathways. This extremely artificial condition is often overlooked in experimental approaches and may bias the results. Furthermore, some molecules in the organoid medium have unpredictable off-target effects that significantly impact the behavior and maturation of certain cell populations. Nicotinamide, gastrin and PGE2 inhibit immune responses. SB202190, A83-01 and vanadate (from advanced DMEM-F12) modify intracellular signaling. N-AcetylCysteine and Primocin modify the redox response and mitochondrial metabolism, respectively. Thus, the unintentional addition of these molecules to the organoid medium introduces biases under specific experimental settings. While the original organoid medium formula is the gold standard for propagating organoids in vitro, more focused, reliable conditions are necessary for specific organoid-based tests.