Kopp et al., 2024

Current pre-clinical drug safety assessments lack a single, comprehensive test system for genotoxicity hazards identification. This study, aimed to develop an in vitro model to address this predictive genotoxicity gap using Organ-on-a-chip (OOC) technology. OOC present robust human metabolic activity, and has the potential to assess all required endpoints for genotoxicity hazards identification, ultimately streamlining the process and eliminating the need for animal testing.

Liver-on-a-chip (LOC) models hold promise as they closely mimic in vivo environments. In this study, primary human hepatocytes (HepaRG), were grown in MPS-T12 (Barrier) plates with human lymphoblastoid (TK6) using Transwell inserts. OOC cultures were maintained under microfluidic flow using the PhysioMimix® Core System. This facilitated cross-communication between compartments and the analysis of three key genotoxicity endpoints:

1. DNA strand breaks (comet assay) in hepatocytes
2. chromosome loss/damage (micronucleus assay) in TK6 cells,
3. mutation (Duplex Sequencing) in TK6 cells

The system’s functional metabolic capabilities were confirmed by assessing cytochrome activity, RNA expression, albumin, urea, and lactate dehydrogenase production.
We then exposed the system to various genotoxicants at different time points (0, 24, and 45 hours)
direct-acting genotoxicants:

• methyl methanesulfonate (MMS)
• ethyl methanesulfonate (EMS)

and genotoxicants requiring metabolic activation:

• benzo[a]pyrene (B[a]P)
• cyclophosphamide (CP)

Genotoxic responses were observed for all endpoints with MMS and EMS. Additionally, increased micronucleus and mutation frequencies were detected with CP, and %Tail DNA increased with B[a]P, indicating the system’s metabolic competence. Interestingly, CP did not induce increased %Tail DNA, aligning with in vivo data. However, B[a]P did not trigger an increase in micronucleus and MF, suggesting potential room for optimization.

In conclusion, this proof-of-concept experiment demonstrates the potential of PhysioMimix Liver-on-a-chip model for in vitro genotoxicity hazards identification, paving the way for a more streamlined and animal-free pre-clinical drug safety assessment process.”