208 Chapter 7 techniques such as capillary electrophoresis-mass spectrometry (CE-MS).34,35 These new techniques could allow for reliable and reproducible metabolic profiles of organoid samples with limited biomass. Another important and limiting factor of organoid work is the use of hydrogels. Hydrogels, are extracellular matrix (ECM) proteins that work as a scaffold, allowing organoids to grow in three dimensions36. These matrices are commonly from tumor origin and present high batch-to-batch variability37. Alternatively to animal-derived undefined matrixes, the biomaterials field is currently moving towards alternatives including decellularized extracellular matrix, synthetic hydrogels and gel-forming recombinant proteins38. It is important to mention that biological hydrogels contain high concentrations of proteins and their presence might interfere with downstream analysis, particularly in proteomics analysis. During the work for this thesis, I observed how correct degradation and removal of the hydrogel prior to downstream analysis is essential to obtain reliable proteomics results. This could be caused by the amount of hydrogel left after removal, which can also impact total protein concentrations and cause variations in normalization. Therefore, it is important to establish robust and standardized protocols for the complete removal of matrixes and to avoid issues derived from the hydrogels. Finally, I would also like to bring the attention to a commonly overlooked limitation of organoid work, namely the economic costs. Organoid culture medium often requires expensive growth factors and cytokines, which are used in large quantities. Moreover, the hydrogels needed to grow organoids in three dimensions are also costly and needed in large amounts. Because of these high costs, the organoid technology is not yet accessible to all research groups. Surprisingly, little information has been published on this topic. While the organoid technology slowly becomes a daily practice in more and more labs, it is still far from being accessible to everyone. There is a clear need for companies and academic labs to work together, towards the development and commercialization of robust, well-defined and inexpensive matrixes to standardize biological research. Regarding the in silico models presented in this thesis, some aspects require further attention. These models are presented as tools to predict in vitro/in vivo responses or behaviors. However, in order to build biologically accurate models, we rely on kinetic parameters from the enzymes in the model. While working on chapter 5, I found that there is a lack of standardized and reliable kinetic parameters of the enzymes involved in the peroxisomal β-oxidation. Most of the studies describing kinetics of the enzymes date back to the 80s
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