Validation of a Novel Cell Culture System to Perform 3D in vitro Cytotoxicity Analyses using Primary Hepatocytes


1. Tim Moeller, BioreclamationIVT
2. Brad Larson, BioTek Instruments, Inc.
3. Grant Cameron, TAP Biosystems
4. Peter Banks, BioTek Instruments, Inc.
Hepatocytes are the primary cell type of the liver providing the majority of the detoxification which increases the potential for cellular dysfunction and death. Though the source of the insult may be caused by several factors, exposure to drugs represents a significant concern warranting FDA guidance on drug-induced liver injury (DILI). In vivo studies are still the gold standard; however, in vitro screening has gained importance for reducing animal exposure, amendable to high-throughput platforms and better equipped to study cellular mechanisms of action.
Typically in vitro screening has incorporated primary hepatocytes cultured in a two dimensional (2D) format where the cells form a monolayer across the bottom of a well. However, when cultured and studied in this fashion they rapidly lose their key functions and de-differentiate over the course of only a few days. The ability to culture, characterize and challenge primary cells in a biomimetic 3D environment enables the performance of longer term studies.
Here we present data demonstrating the differences in response between human primary cells cultured in 2D and in the RAFT 3D cell culture system which has the benefits of a collagen hydrogel with tissue-like properties. Camptothecin and pyocyanin were tested for their ability to cause short-term oxidative stress, as well as long-term induction of apoptosis and necrosis. Kinetic live cell imaging was performed using multiple fluorescent non-perturbing probes to monitor the various effects in real time with incubations up to 48 hours which enabled a thorough assessment of the toxin profile. Image overlay allowed for discrete cellular analysis. Variations in cytotoxicity levels were observed after a 24 hour 800 nM camptothecin treatment (75%:2D; 28%:3D). ROS induction was less in 3D than 2D system. Overall, cells exhibited greater viability using the RAFT system, and were less sensitive to toxins than observed in traditional 2D culture for the endpoints which may indicate a more robust cell culture system.

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