Cell Culture


Table 1: 3D primary human hepatocyte spheroids hepatotoxicity assay shows 2-3X improvement in sensitivity versus


2D. IC50 values measured from 2D and 3D hepatotoxicity assays are corrected with clinical Cmax (not shown) for 100 tested drugs that belong to five DILI severity categories1. To compare the performance for 2D and 3D liver spheroids, assay sensitivity and specificity are calculated using MOS thresholds 10X, 25X, and 50X, respectively


spheroid culture on a panel of 123 drugs with and without causative involvement in clinical DILI events2. The 3D model achieved 69% sensitivity and 100% specificity. The authors concluded that their spheroid system “exceeded both the sensitivity and the specificity of all previously published in vitro assays at substantially lower exposure levels.”


Applying 3D to Induced Pluripotent Stem Cells (iPSCs) The convergence of 3D cell culture systems and the ability to culture HiPSCs into functional cell types and tissues creates additional advantages for dis- ease modelling, target identification and lead opti- misation. HiPSCs can be developed by genetic reprogram-


ming of somatic cells such as skin fibroblasts to an embryonic-like state by viral introduction of tran- scription factors that induce pluripotency. The resulting iPSCs are self-renewable and can be directed to differentiate into a variety of cell types. HiPSCs exhibit disease phenotypes close to the human pathology, particularly when cultured under conditions that allow them to recapitulate tissue architecture in the form of multicellular spheroids or organoids. When cultured under 3D conditions, human


iPSCs clearly provide optimised systems that more accurately reflect disease-related target mutations, compound pharmacology and toxicology. HiPSCs and 3D cell culture systems can also be used to generate complex, multicellular systems in which cells are spatially arranged similarly to tissues in vivo. These organoids and more complex organ systems are being leveraged for lead optimisation, including estimations of preclinical toxicity and potential metabolic liability.


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Below, we explore the use of HiPSC-derived 3D liver tissue for drug discovery and lead optimisation.


3D HiPSC-derived liver tissue Drug discovery HiPSC-derived human hepatocytes are being employed in drug discovery against molecular tar- gets that cause liver disease. For example, HiPSC- derived hepatocytes generated from patients with homozygous familial hypercholesterolemia enabled identification of compounds that lower serum low- density lipoprotein C (LDL-C). Cayo et al have identified several cardiac glycosides that lowered expression of apolipoprotein B in the human hepa- tocytes in vitro3. Patients treated with these same cardiac glycosides also had reduced serum LDL-C levels, collectively supporting the potential use of cardiac glycosides to treat hypercholesterolemia.


Lead optimisation – toxicity evaluation Primary human hepatocytes can be used to profile compounds for their potential to induce liver toxicity and for their metabolic liability in patients. Because human hepatocytes may respond differently to drugs than rodent- or tumour-derived hepatocytes, their use provides major advantages in testing for drug safety at the preclinical discovery stage. However, the source of these cells is limited, restricting their widespread use in drug safety screens. In contrast, human hepatocytes derived from


HiPSCs can be produced in high abundance and reproducibility, with the desired genetic profile. 3D cell culture systems developed using HiPSC- derived hepatocytes offer significant value in eval- uating the potential of drugs for toxicity. Liver cells grown in organoid culture have advantages over cells grown in 2D as they develop


Drug Discovery World Winter 2018/19


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