206 Chapter 7 During the course of the PhD, I have encountered multiple challenges in the establishment and application of metabolic techniques onto organoid cultures. Most of these issues stemmed from either low reproducibility between organoid cultures, low number of cells, or the presence of hydrogels such as Matrigel or BME (Figure 1). Figure 1. Technical limitations of organoids cultures in metabolic research and proposed approaches to overcome them. Created with BioRender.com One relevant point to discuss is the high variability observed between organoid cultures. It is well known that organoids are highly heterogenous, displaying variability between cells within the same organoid (intra-organoid heterogeneity), between organoids within the same plate and between organoids from different patients (inter-organoid heterogeneity)16. While this heterogeneity is crucial to capture differences between patients, and therefore for personalized-medicine purposes, it is also a setback in reproducibility16,31. While inter-organoid heterogeneity is not a problem, inter-organoid variability can minimize reproducibility in many downstream analyses. When working on metabolic profiling of these organoids, it is common to observe disparate absolute values of metabolites within different organoid cultures when they
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