Biotransformation of diazepam in a clinically relevant flat membrane bioreactor model using primary porcine hepatocytes

Abstract

In vitro biotransformation of drug using commercial culture medium with serum may not be the ideal culture medium for clinical application in extracorporeal bioartificial liver support (BAL) systems. In these systems, patient's blood or plasma is plumbed to primary hepatocytes within a seeded bioreactor, creating interaction between plasma and seeded hepatocytes. To address this situation, we investigated the biotransformation potential of diazepam in primary porcine hepatocytes with a flat membrane bioreactor (FMB); we used human plasma exposure and serum-free media in organotypical double gel culture model for long-term culture. We investigated diazepam clearance and all major metabolites of diazepam, such as oxazepam, temazepam, and desmethyldiazepam, in conventional single gel and organotypical sandwich models and compared them to the FMB model. Diazepam elimination was higher in double gel cultures with exposure to both SF 3 medium conditions and plasma, when compared to the single gel model in a Petri dish. It was observed that in the FMB, diazepam elimination was stable at about 3 pg/h/cell in plasma and SF 3 exposure. Oxazepam synthesis in the bioreactor was approximately one quarter less than in the Petri dish, but there were no differences between N-desmethyldiazepam and temazepam synthesis in double gel culture. In the flat membrane bioreactor, there was no decrease in the biotransformation of diazepam in plasma exposure compared with the control group. Our results suggest that this plasma exposure bioreactor may offer a useful approach in clinical use of extracorporeal BAL, as well as for drug metabolite investigation into toxicological research.

Profiling the Impact of Medium Formulation on Morphology and Functionality of Primary Hepatocytes in vitro

“…Both M199 and M1640 show differential effects on urea synthesis capacity, the latter significantly greater than all media tested (Fig. 1), and substantially higher than previously published reports631. Total CYP450 content remained stable in 2-day-old cultures, despite a 66% decrease compared with fresh cells although ML-15 maintained day 2 tCYP450 levels up to culture day 4.…”

“…The importance of identifying a culture medium which promotes hepatotrophic support, viability and differentiated function in vitro for BALs, is well recognised6203239. The aim of this study was to assess in vitro retention of synthetic, detoxification and metabolic parameters of high-density hepatocytes cultured in fully defined WE, Modified L-15, M199 and RPMI 1640 media formulations; integrate these datasets using the HBAI to assess global functional capacity, with parallel morphological parameters to elucidate hepatotrophic support suitability.…”

“…Recent insights also support the use of PPHs as authentic surrogates for PHHs, given their similar phenotype and function456. Availability of PHHs for cell-based approaches to treat acute liver failure (ALF) is severely restricted due to a shortage of donor organs.…”

Detection of nanolevel drug metabolites in an organotypic culture of primary human hepatocytes and porcine hepatocytes with special reference to a two-compartment model

“…liver features, such as cuboidal morphology, bile canaliculi, tight junctions, and gap junctions. 39,40 Advanced organotypic cellular models provide challenges in the biotransformation of drugs; the conventional in vitro model is highly criticized. Research (in both the pharmacological and toxicological fields) into the expression of drug-metabolizing enzymes also relies on cell culture models during the biotransformation of drugs.…”

Arginine–glycine–aspartic acid–polyethylene glycol–polyamidoamine dendrimer conjugate improves liver-cell aggregation and function in 3-D spheroid culture

“…Commonly used methods are coculture with other cells, 2 microencapsulated culture, 3 spheroidal aggregate culture, 4 and bioreactor culture. 5 Spheroidal aggregate culture makes hepatic cells aggregate into a sphere, in which the contact area is the largest. This phenomenon leads to the formation of a cube morphology and cytoskeleton structure similar to in vivo and simulates the microenvironment in vivo.…”