Presentation Information
[P01-153]Optogenetic control of hepatic function in engineered hepatoma cells for toxicity assessment
○Sho Shimokado1, Keigo Honda, Feiyang Zheng1, Silas Habimana1, Nana Shirakigawa1, Yoshinori Kawabe1, Masamichi Kamihira1 (1. Kyushu University (Japan))
Keywords:
Optogenetics,light-inducible gene expression,engineered hepatoma cells,hepatic function,hepatotoxicity screening
[Purpose]
Synthetic biology enables precise control of cellular functions through gene circuit engineering. We previously established HepG2/8F_HS, an engineered hepatoma cell line for liver modeling and drug evaluation. This system employs a Tet-based activation system in which thermal induction triggers tTA expression, thereby switching cells from a proliferative state to a functionally mature state. Although heat induction is effective, light-mediated induction offers greater convenience and superior spatiotemporal control. In this study, we developed a hepatoma cell line harboring a light-inducible gene circuit based on the CRY2/CIB1 light-dependent interaction derived from Arabidopsis thaliana and evaluated its applicability for light-induced hepatotoxicity assessment.
[Method]
The pBlueL-TA vector, designed to co-express TetR/CIB1 and CRY2/p65, was integrated into the HepG2/8F_HS genome using a transposon-based lipofection system to generate the light-switchable HepG2/8F_LS cell population. A stable monoclonal cell line, HepG2/8F_LS #8, was established by limiting dilution. Thermal induction was performed at 43°C for 30 min. Light irradiation (460 nm, 160,000 lux) was applied using various pulsed irradiation protocols over a total duration of 3 h.
[Results]
Three days after thermal induction, parental HepG2/8F_HS cells exhibited an albumin secretion rate of 16.0 pg/(cell·day) and an ammonia removal rate of 0.193 pmol/(cell·h). The HepG2/8F_LS #8 clone also demonstrated comparable functional capacity under thermal stimulation, with an albumin secretion rate of 12.1 pg/(cell·day) and an ammonia removal rate of 0.189 pmol/(cell·h). When HepG2/8F_LS #8 cells were subjected to a pulsed light irradiation protocol (1 s ON / 29 s OFF for 3 h), the GFP-positive rate reached 70.0% after 24 h. Three days after light exposure, the cells exhibited an albumin secretion rate of 11.6 pg/(cell·day) and an ammonia removal rate of 0.132 pmol/(cell·h). The highest induction efficiency (GFP-positive rate of 91.1%) was achieved using a pulse condition of 3 s ON / 27 s OFF within a fixed 30-s cycle over a total duration of 3 h.
[Discussion]
HepG2/8F_LS #8 retained thermal induction capacity comparable to that of the original HS line. Among the tested conditions, the 3s ON / 27s OFF protocol (total exposure of 18 min) was the most effective for functional induction. Further optimization of parameters, such as total irradiation duration, may enhance induction efficiency. Transition from thermal to light-dependent induction provides a more streamlined and less invasive platform for regulating hepatic function.
[Conclusion]
By introducing light irradiation as an induction factor, we established a high-precision platform for regulating liver function. Owing to its convenient controllability, this system is expected to serve as a powerful tool for advancing high-throughput hepatotoxicity screening and drug safety assessment in pharmaceutical research.
Synthetic biology enables precise control of cellular functions through gene circuit engineering. We previously established HepG2/8F_HS, an engineered hepatoma cell line for liver modeling and drug evaluation. This system employs a Tet-based activation system in which thermal induction triggers tTA expression, thereby switching cells from a proliferative state to a functionally mature state. Although heat induction is effective, light-mediated induction offers greater convenience and superior spatiotemporal control. In this study, we developed a hepatoma cell line harboring a light-inducible gene circuit based on the CRY2/CIB1 light-dependent interaction derived from Arabidopsis thaliana and evaluated its applicability for light-induced hepatotoxicity assessment.
[Method]
The pBlueL-TA vector, designed to co-express TetR/CIB1 and CRY2/p65, was integrated into the HepG2/8F_HS genome using a transposon-based lipofection system to generate the light-switchable HepG2/8F_LS cell population. A stable monoclonal cell line, HepG2/8F_LS #8, was established by limiting dilution. Thermal induction was performed at 43°C for 30 min. Light irradiation (460 nm, 160,000 lux) was applied using various pulsed irradiation protocols over a total duration of 3 h.
[Results]
Three days after thermal induction, parental HepG2/8F_HS cells exhibited an albumin secretion rate of 16.0 pg/(cell·day) and an ammonia removal rate of 0.193 pmol/(cell·h). The HepG2/8F_LS #8 clone also demonstrated comparable functional capacity under thermal stimulation, with an albumin secretion rate of 12.1 pg/(cell·day) and an ammonia removal rate of 0.189 pmol/(cell·h). When HepG2/8F_LS #8 cells were subjected to a pulsed light irradiation protocol (1 s ON / 29 s OFF for 3 h), the GFP-positive rate reached 70.0% after 24 h. Three days after light exposure, the cells exhibited an albumin secretion rate of 11.6 pg/(cell·day) and an ammonia removal rate of 0.132 pmol/(cell·h). The highest induction efficiency (GFP-positive rate of 91.1%) was achieved using a pulse condition of 3 s ON / 27 s OFF within a fixed 30-s cycle over a total duration of 3 h.
[Discussion]
HepG2/8F_LS #8 retained thermal induction capacity comparable to that of the original HS line. Among the tested conditions, the 3s ON / 27s OFF protocol (total exposure of 18 min) was the most effective for functional induction. Further optimization of parameters, such as total irradiation duration, may enhance induction efficiency. Transition from thermal to light-dependent induction provides a more streamlined and less invasive platform for regulating hepatic function.
[Conclusion]
By introducing light irradiation as an induction factor, we established a high-precision platform for regulating liver function. Owing to its convenient controllability, this system is expected to serve as a powerful tool for advancing high-throughput hepatotoxicity screening and drug safety assessment in pharmaceutical research.
Comment
To browse or post comments, you must log in.Log in