Abstract
A critical requirement for assessing drug toxicity in vitro is cytochrome P450 (CYP) function that is physiologically representative. Due to low levels of CYP enzyme activity, cell lines such as HepG2 hepatocytes have largely been supplanted with primary cells derived from human donor tissue. While these cells retain CYP activity ex vivo, drug screening is limited to short-term assessments due to de-differentiation. This limits their efficacy in testing slow-clearing, large-molecule compounds such as antibody-based therapies and low-turnover drugs. Cell lines in monolayer cultures have been effective in assessing the safety of small molecule drugs, but the disparity between cell lines and in vivo tissue biology proves too substantial for current potential therapies. Three-dimensional (3D) cultures have been shown to be more physiologically relevant models of in vivo tissue. However, even they fail to fully mimic living tissue and suffer from the lack of active oxygen and nutrient transport. Lena Biosciences has developed a groundbreaking in vitro system that facilitates long-term survival of 3D cultures. Cells seeded in 3D hydrogels supported by a rigid scaffold are maintained in statistically independent wells in their own medium which floats atop a high-density blood-substitute in which medium is immiscible. Infusion and withdrawal of this blood-substitute introduces perfusion into a medium-throughput multiwell system without the need for separate pumps for each culture condition.
Internal validation experiments using hepatocytes cultured in a 12-well system showed a two-fold improvement in cellular respiration (measured by alamarBlue assay) over cultures maintained in a standard multiwell plate when the blood-substitute was introduced, and a three-fold improvement when the cultures were perfused. Cell viability (measured by LDH release) was significantly improved when perfusion was introduced. CYP activity (assessed using both fluorescent and luminescent substrate-based assays) was also significantly higher in the perfused hepatocyte cultures. These data demonstrate the substantial advantages to growing cells in this novel perfused cell culture model. Scaling the system to include multiple cell types (primary or cell lines), would allow for simultaneous testing of target and off-target effects of potential small or large molecule therapies. Thus, this system serves as a major improvement over the current organ-on-a-chip solutions in the area of drug safety testing.
Citation Format: James T. Shoemaker, Jelena Vukasinovic. Cytochrome P450 enzyme activity is enhanced in hepatocytes grown using a perfused 3D cell culture drug screening system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4080. doi:10.1158/1538-7445.AM2017-4080
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