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Cell Culture


relevant cell-cell interaction due to the polarity of the hepatocytes. This polarity is critical for cell metabolism which, in turn, is a feature essential for drug metabolism. Most importantly, human liver organoids respond differently to drugs than similar cells grown in 2D culture. Because of differences in expression of


metabolising P450 enzymes between individuals, one can envision drugs screened against large pop- ulations of HiPSC-derived hepatocytes in 3D to provide a better preclinical assessment of the potential to induce toxicity than presently avail- able with any other in vitro screening system. Compound screening of potentially hepatotoxic


compounds against HiPSC-derived hepatocytes have yielded similar results as when the same compounds were tested against primary human hepatocytes. These data and others validated the use of


HiPSC-derived hepatocytes for preclinical lead optimisation and showed that the cells had a high degree of clinical predictability.


Lead optimisation – compound metabolism screening Genetic variability may cause differences from one individual to another in P450 enzyme expression, which can result in individuals being either ‘slow’ or ‘fast’ metabolisers of many commonly pre- scribed drugs. HiPSC-derived hepatocytes can be used to evaluate the metabolic liability of com- pounds and have the potential to be used to assess the influence of patient genetic variability on drug metabolism. The utility of the HiPSC-derived hepatocytes in


this regard was reported by Takayama et al, who used HiPSC-derived hepatocytes isolated from indi- viduals with or without a single-nucleotide poly- morphism (SNP) in the P450 enzyme, CYP2D64. The SNP in the CYP2D6 genes reduces enzymatic activity and individuals with this SNP are slow metabolisers of drugs, including tamoxifen. Normally, CYP2D6 metabolises tamoxifen to a


cytotoxic metabolite in breast cancer cells. Co-cul- tures of MCF7 human breast cancer cells with HiPSC-derived hepatocytes from individuals with the normal CYP2D6 enzyme showed substantial loss of cancer cells after tamoxifen treatment. As expected, cancer cells co-cultured with hepatocytes from individuals with the CYP2D6 SNP showed resistance to tamoxifen, because less of the cyto- toxic metabolite was produced, and ultimately diminished breast cancer cell toxicity. Humans vary in the expression levels of P450


enzymes, and therefore metabolise drugs different- ly. HiPSC-derived hepatocytes therefore provide


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better models of the diversity of metabolic responses existing in patient populations. Therefore, they may provide better predictors of clinical efficacy and safety than many other mod- els of liver function. These properties of HiPSC-derived hepatocytes


may also facilitate the development of unique multi-tissue/organ systems to determine human drug toxicity. End-organ toxicity is a major limit- ing factor in drug development. Consequently, approaches that predict preclinical drug safety may provide the means to reduce the attrition rate of novel leads in the clinic. In this respect, microfluidic systems have proven to be very use- ful. Here, different organoids derived from HiPSCs – such as hepatocytes, cardiac myocytes, gastrointestinal and kidney cells or neurons – are grown in separate, but interconnected, chambers, and can be used to profile drugs and their metabo- lites simultaneously for potential toxicity in differ- ent tissues in the body5.


The way forward While 3D cell cultures present many advantages in drug discovery, hurdles remain before their widespread adoption can be achieved. Our recent survey highlighted key areas which must be addressed. The first is the need for advances in imaging and detection techniques to effectively monitor tissues in 3D cell culture. Despite increasing adoption of 3D approaches,


challenges remain as to the way spheroid cultures are characterised and analysed. This analysis is particularly important when screening compounds in spheroid culture in which cells can organise into a proliferating zone, a quiescent region of viable cells and a hypoxic, necrotic core. All regions of the spheroid must be analysed to properly assess the impact of drug candidates. The thickness of 3D cell cultures (typically


>100m) causes light scattering and limits imaging to the surface-layer cells, preventing complete char- acterisation of the cell population within whole- mount 3D cell cultures. This introduces a sampling bias in imaging analysis, since only the exterior cells can be imaged where concentrations of nutri- ents, oxygen and drug compound are greatest. While the high cellular density of 3D cell cul-


tures scatters light and limits the ability to charac- terise the entire spheroid, several approaches are available for rendering tissue transparent to enable analysis. Commonly-used clearing techniques have a number of disadvantages when being considered for high throughput, cost-efficient screening, how- ever. Protein hyperhydration can require days to


Drug Discovery World Winter 2018/19


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