Simultaneous quantitative measurement of luciferase reporter activity and cell number in two- and three-dimensional cultures of hepatitis C virus replicons.
Hepatitis C virus (HCV) is a global health problem and an important human pathogen. The development of cell culture models for HCV infection has been difficult to accomplish, primarily because HCV is very sensitive to the host cell state. Future models will require the use of three-dimensional (3D) cultures that model the host cell state and environment more accurately. Higher information content screens for anti-HCV therapeutics will also involve 3D cell cultures. Here we report a method for screening cell models for HCV replication that involves normalizing luciferase reporter activity based on cell number in two-dimensional (2D) and 3D HCV replicon cultures. Human hepatoma cells stably replicating luciferase-containing HCV replicons were cultured in 2D monolayer culture and 3D spheroid culture. Optimization of cell lysis was performed so that cell lysates could be used to quantify both luciferase activity and cellular DNA content. Cellular DNA content was quantified using Hoechst 33258 dye and was converted to cell number. The method is straightforward, reproducible, and sensitive down to 5000 cells. This method enables low-throughput but high-information content screening of HCV replicons, with the potential for high-throughput screening in a variety of 3D cultures and cocultures.
Tonary
Angela M
AM
Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, Ont., Canada K1A 0R6.
Pezacki
John Paul
JP
D006690:Bisbenzimidazole / Q000737:chemistry*; D006528:Carcinoma, Hepatocellular; D002452:Cell Count / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000032:analysis; D017930:Genes, Reporter / Q000235:genetics*; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000235:genetics*; D012093:Replicon / Q000235:genetics* / Q000502:physiology; D015203:Reproducibility of Results
D004279:DNA, Viral; D008156:Luciferases; D006690:Bisbenzimidazole