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Exploration and application of a liver-on-a-chip device in combination with modelling and simulation for quantitative drug metabolism studies
Filed under: ADME and Drug metabolism
Summary
Researchers at Roche and academic partners used the CN Bio PhysioMimix® liver-on-a-chip, seeded with single-donor primary human hepatocytes in LC-12 plates, to measure drug metabolism for 15 compounds and to extract quantitative drug metabolism and pharmacokinetics (DMPK) parameters with mathematical modelling. The liver-on-a-chip measured intrinsic clearance (CL_int) for cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and aldehyde oxidase (AO) substrates, estimated the fraction metabolized (f_m) for quinidine and diclofenac, and detected all efavirenz metabolites reported in vivo, as well as secondary and tertiary metabolites. Perfused culture supported undisturbed incubations for up to 96 hours, and an evaporation-corrected model yielded more accurate CL_int values for low-clearance drugs, correcting an underestimation of up to 40% compared with a simple linear analysis. The work matters because it shows that the liver-on-a-chip, combined with cell-number normalization, evaporation correction, and appropriate model selection, generates reliable clearance, f_m, and metabolite data, including for metabolically stable drugs that standard short-incubation systems struggle to characterize.
Study facts at a glance
| Publication | Docci L, Milani N, Ramp T, Romeo AA, Godoy P, Ortiz Franyuti D, Krähenbühl S, Gertz M, Galetin A, Parrott N, Fowler S. Exploration and application of a liver-on-a-chip device in combination with modelling and simulation for quantitative drug metabolism studies. Lab Chip. 2022;22:1187-1205. |
| DOI | 10.1039/d1lc01161h |
| CN Bio product used | PhysioMimix® LC12 and PhysioMimix Core System |
| How the platform was used | Single-donor primary human hepatocytes were cultured under continuous medium perfusion in LC-12 plates (1,800 μL accessible medium per well) for up to 96 hours, and serial medium sampling with liquid chromatography tandem mass spectrometry (LC-MS/MS) measured drug depletion and metabolite formation for intrinsic clearance, fraction metabolized, and metabolite identification. |
| Biological context | Human hepatic drug metabolism, studied with healthy single-donor primary human hepatocytes (lot Hu8264) in the liver-on-a-chip; a drug metabolism and pharmacokinetics (DMPK) study rather than a disease model. |
| Comparator | Suspended human hepatocytes and HepatoPac micropatterned co-cultured hepatocytes (intrinsic clearance), observed clinical clearance (in vitro to in vivo extrapolation), and a linear model versus an evaporation-corrected model. |
| Key readouts | Hepatocyte seeding efficiency and active cell number (total protein, albumin production rate), intrinsic clearance (CL_int) from drug depletion, in vitro to in vivo extrapolation (IVIVE) of hepatic clearance, fraction metabolized (f_m) for quinidine and diclofenac, and efavirenz metabolite identification. |
| Main interpretation | Combining the liver-on-a-chip with mathematical modeling produces quantitative DMPK parameters with reasonable human translatability, and long perfused incubations with evaporation correction extend reliable intrinsic clearance estimates to low-clearance drugs. |
Table of Contents
Which CN Bio product was used?
The study used the CN Bio PhysioMimix liver-on-a-chip, microfluidic microphysiological system that perfuses culture medium across cultured hepatocytes, supplying nutrients, oxygen, and mechanical stimulation via shear stress, along with Liver Chip-12 consumable plates and the CN Bio controller, docking station, and drivers. Single-donor cryopreserved primary human hepatocytes (lot Hu8264) were seeded on the LC-12 plate scaffolds at between 400,000 and 650,000 cells per well (consensus 600,000) and pre-incubated for around 72 hours before treatment. Each well contained an accessible medium volume of 1,800 μL, allowing repeated medium sampling from the same well during incubations of up to 96 hours and enabling the construction of an in vitro pharmacokinetic profile for each well. The platform was central to the drug metabolism measurements, while separate assays (equilibrium dialysis for protein binding, LC-MS/MS for drug quantification) and in silico modeling supported the analysis.
What this paper is about
Standard in vitro systems for drug metabolism, including 2D plated hepatocytes, suspended hepatocytes, and liver microsomes, rapidly lose enzyme activity and yield poor clearance estimates for metabolically stable, low-clearance drugs. That limits quantitative prediction of human pharmacokinetics during candidate selection. This study evaluates whether the PhysioMimix liver-on-a-chip, which keeps hepatocytes under continuous perfusion, meets the minimum requirements for DMPK use and can support more demanding endpoints. It first develops the cell-number normalization and evaporation-correction methods, then tests the system across three application areas: intrinsic clearance and IVIVE for 15 drugs, fraction metabolized for quinidine and diclofenac, and metabolite identification for efavirenz. Mathematical modeling was used throughout for experiment planning and data analysis.
What the researchers found
Seeding and cell number: seeding efficiency averaged 52% (range 39 to 66.5%), leaving around 260,000 active hepatocytes per well at day 2. Albumin production rate (39 ± 8 μg per day per million cells), at the lower end of the range estimated for human liver in vivo and above rates recently reported for hepatocyte spheroids, worked as a non-invasive marker to estimate the active cell number in each well, correcting for well-to-well differences.
Intrinsic clearance: CL_int was measured for four CYP substrates, seven UGT substrates, and one AO substrate, with low inter-well variability in calculated cell number (mean coefficient of variation 12%). Values fell within 2-fold of suspended hepatocytes for 11 of 14 compounds and within 2-fold of HepatoPac for 8 of 13 compounds, which the authors describe as promising for a new single-donor system compared against highly optimized pooled hepatocyte systems.
Low-clearance drugs: undisturbed 96-hour incubations allowed CL_int measurement for metabolically stable drugs. For low-clearance drugs (CL_int below 1 μL per min per million cells) with evaporation rates above 0.05 μL/min, an evaporation model that accounted for medium loss corrected the underestimation of up to around 40% seen with a simple linear model, as shown for tolbutamide and irinotecan.
IVIVE: predicted hepatic intrinsic clearance was within 3-fold of observed values for 7 of 12 compounds (midazolam, quinidine, diclofenac, naloxone, oxazepam, tolbutamide, and lorazepam), with satisfactory prediction for permeable, metabolically cleared compounds (extended clearance classification system classes 1a and 2) and no difference in prediction success between CYP and UGT substrates. The remaining compounds were under-predicted; for the hepatic uptake transporter substrates telmisartan and repaglinide, this matches trends seen in other closed in vitro systems, and the authors identify bile flow capability and pooled multi-donor hepatocytes as next steps.
Fraction metabolized: for diclofenac, the model showed a fraction metabolized by UGT (f_m,UGT) of 0.64, close to a literature value of 0.62 from human liver microsomes, in what the authors report as the first use of the PhysioMimix liver-on-a-chip with modeling to determine the UGT contribution to diclofenac metabolism. For quinidine, the 3-hydroxyquinidine pathway f_m was estimated at 0.64 to 0.71 depending on model structure.
Metabolite identification: all four efavirenz metabolites reported in vivo were detected in the liver-on-a-chip, along with secondary and tertiary metabolites, indicating the system can support metabolite identification for low-turnover drugs. The authors attribute this to the large medium volume and to cell longevity, which allows metabolites with low formation rates to be detected after extended incubation.
Sustained metabolic activity: undisturbed incubations of lorazepam, posaconazole, and tolbutamide showed linear depletion profiles over 4 days, with no indication of substantive loss of enzyme activity, in contrast to the 40 to 50% loss within 24 hours of seeding reported for 2D monocultured hepatocytes. Under a repeated-use protocol with daily medium replacement, which the authors describe as a worst-case scenario, more than 80% of metabolic activity was retained at day 2 for all seven substrates tested.
Why the paper matters
The paper provides a detailed methodology for deriving quantitative DMPK parameters from perfused liver-on-a-chip data, rather than treating the device as a black box. It shows which experimental and modeling steps are needed: normalizing to active cell number using albumin, correcting for medium evaporation during long, low-clearance incubations, and selecting model structures for the fraction metabolized. Because perfused hepatocytes retain metabolic activity over extended incubations, the liver-on-a-chip enables endpoints that standard short-incubation systems struggle with for metabolically stable drugs, including low intrinsic clearance, f_m, and metabolite identification. For drug metabolism scientists, this provides a clear basis for using the liver-on-a-chip to inform candidate selection and human dose prediction, with data that already show reasonable human translatability.
Key study takeaways
- The study used the CN Bio PhysioMimix liver-on-a-chip with LC-12 plates and single-donor primary human hepatocytes to measure drug metabolism for 15 compounds.
- Albumin production rate (39 ± 8 μg per day per million cells) worked as a non-invasive marker for active hepatocyte number, correcting for a seeding efficiency that averaged 52%.
- Intrinsic clearance from the liver-on-a-chip fell within 2-fold of suspended hepatocytes for 11 of 14 compounds and, after in vitro-to-in vivo extrapolation, within 3-fold of observed human values for 7 of 12 compounds.
- Undisturbed incubations of up to 96 hours, combined with an evaporation-corrected model, gave more accurate intrinsic clearance values for low-clearance drugs such as tolbutamide and irinotecan, correcting an underestimation of up to around 40%.
- The system estimated a diclofenac fraction metabolized by UGT (f_m,UGT) of 0.64, close to the human liver microsome value of 0.62, and detected all four efavirenz metabolites reported in vivo, plus secondary and tertiary metabolites.
- Perfused hepatocytes retained more than 80% of metabolic activity at day 2 even with daily medium replacement, and showed linear depletion profiles over 4 days in undisturbed culture, supporting long incubations and repeated measurements from the same cells.
Why this paper is worth reading
This paper is useful because it sets out, step by step, how to generate trustworthy quantitative data from a perfused liver-on-a-chip. A drug metabolism scientist deciding whether to adopt the PhysioMimix liver-on-a-chip will find concrete guidance on normalizing to active cell count, correcting for evaporation during long, low-clearance incubations, and selecting model structures for fraction metabolized. The paper also shows the platform delivering clearance, f_m, and metabolite-identification data for metabolically stable drugs, providing teams with quantitative inputs for candidate selection and drug-drug interaction risk assessment.
FAQ
The study used the CN Bio PhysioMimix liver-on-a-chip with LC-12 plates.
Single-donor primary human hepatocytes were seeded on the LC-12 plate scaffolds and cultured under continuous medium perfusion, with an accessible medium volume of 1800 μL per well. Serial medium samples were taken over incubations of up to 96 hours and analyzed by LC-MS/MS to measure drug depletion and metabolite formation.
The study modeled human hepatic drug metabolism using primary human hepatocytes in the liver-on-a-chip. It focused on drug metabolism and pharmacokinetics rather than a specific disease, using healthy single-donor hepatocytes.
The PhysioMimix liver-on-a-chip, combined with mathematical modelling, produced quantitative intrinsic clearance, fraction metabolized, and metabolite identification data. Correcting for medium evaporation prevented intrinsic clearance underestimation of up to 40% for low-clearance drugs.
Liver-on-a-chip intrinsic clearance was compared with suspended hepatocytes and HepatoPac micropatterned co-cultures, and predicted in vivo clearance was compared with observed clinical clearance. A linear model was compared with an evaporation-corrected model.
The main readouts were hepatocyte seeding efficiency and active cell number (total protein and albumin production rate), intrinsic clearance from drug depletion, in vitro to in vivo extrapolation of hepatic clearance, fraction metabolized for quinidine and diclofenac, and efavirenz metabolite identification.
The paper gives drug metabolism scientists a practical methodology for producing quantitative DMPK parameters from a perfused liver-on-a-chip, including cell-number normalization, evaporation correction, and model selection for fraction metabolized. It also states where the system is currently limited, such as transporter-mediated clearance.
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