Recursos Técnicos - Posters Científicos
Validation of a Novel 3D Cell Culture System to Perform in vitro Cytotoxicity Analyses using Human Primary HepatocytesDescargar
January 21, 2014
Authors: Brad Larson and Peter Banks, BioTek Instruments, Inc., Winooski, Vermont, USA; Grant Cameron, TAP Biosystems, Royston, Hertfordshire, UK; Timothy Moeller, BioreclamationIVT, Baltimore, Maryland, USA
Hepatocytes are the primary cell type of the liver providing the majority of the detoxification which may increase 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, being amendable to higher-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 microplate 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 longer term studies with more relevant activities.
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 benefi ts 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 induction of apoptosis and eventual cytotoxicity. Kinetic live cell imaging was performed using multiple fluorescent nonperturbing probes to monitor the various effects in real time with incubations up to 24 hours which enabled a thorough assessment of the toxin profile. Image overlay allowed for discrete cellular analysis. Overall, cell health was greater using the RAFT system after a 14 day incubation period (100%:3D vs. 60%:2D cell viability and 100%:3D vs. 65%:2D CYP3A4 activity). Hepatocytes cultured in 3D were also less sensitive to toxins than observed in traditional 2D culture, as exhibited by the lack of immediate reactive oxygen species induction in 3D, as well as variations in observed cytotoxicity levels over time with multiple camptothecin treatments (52%:2D vs. 7%:3D following 3 days at 20 μM; and 100%:2D vs. 39%:3D following 7 days at 1 μM), which may indicate a more robust, biologically relevant cell culture system.