P146 DEVELOPMENT OF A PRIMARY HUMAN HEPATOCYTE SPHEROID CULTURE MODEL FOR DRUG METABOLISM AND DISPOSITION STUDIES

Kajsa P Kanebratt , Cardiovascular & Metabolic Diseases iMed DMPK, AstraZeneca R&D Gothenburg, Mölndal, Sweden
Annika Janefeldt , DMPK, Cardiovascular and metabolic diseases, AstraZeneca R&D Gothenburg, Mölndal, Sweden
Alexandra Peric , AstraZeneca R&D Gothenburg, Mölndal, Sweden
Julia Jonsson , AstraZeneca R&D Gothenburg, Mölndal, Sweden
Linnea Johansson , AstraZeneca R&D Gothenburg, Mölndal, Sweden
Constanze Hilgendorf , DMPK and Bioanalytical Chemistry, AstraZeneca R&D Gothenburg, Mölndal, Sweden
Tommy B Andersson , DMPK, Cardiovascular and metabolic diseases, AstraZeneca R&D Gothenburg, Mölndal, Sweden
Complex processes in the liver such as drug uptake, metabolism, and efflux is difficult to study using established cell suspension or monolayer cultures since primary human hepatocytes are rapidly de-differentiating in these systems. This is especially true for drugs that are dependent on specific receptors for uptake, slowly metabolized or dependent on transporters for cell efflux. To address this, we developed 3D spheroid cultures using primary human hepatocytes alone or in co-culture with primary human stellate cells. Cells were seeded in ultra-low attachment plates at a density of 2000 cells/well and spheroids were fully formed after five to seven days. Spheroids were sampled at different time points over a three week culture period and hematoxylin and eosin staining did not show any evidence of necrotic core in the spheroids during the whole culture period. Immunohistochemical analysis also revealed staining of the drug metabolizing enzyme CYP3A4 and the drug transporter MRP2 throughout the spheroid. The expression and activities of the major drug metabolizing cytochrome P450 enzymes were retained throughout the whole culturing period of three weeks indicating a stable functionality over time. The activity of the bile canalicular transporter MRP2 was also investigated using the fluorescent substrate CMFDA. The MRP2 transporter was found to be active over the culture time and could be inhibited by the selective MRP2 inhibitor probenecid. Thus, the liver spheroid 3D culture model shows promising results for long term metabolism and disposition studies and has the potential to be developed into a disease model and used in targeted drug delivery studies.