Application Note
Modeling the Liver-Brain Axis with NANOSTACKS™
A connected multi-organ model of the liver-brain axis for studying metabolite-driven neurotoxicity, first-pass metabolism, and systemic drug distribution.
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Overview
Many drug-induced adverse effects arise from multi-organ interactions — particularly the sequential processing of a compound by the liver followed by distribution to secondary target organs. Hepatic metabolism generates reactive metabolites or bioactive transformation products that may be toxic to the brain at concentrations below the parent compound's neurotoxicity threshold.
This application note describes the development of a connected liver-brain axis system using two NANOSTACKS™ organ modules in series. The model enables study of metabolite-driven neurotoxicity, first-pass metabolic effects, and systemic compound distribution — a step towards physiologically relevant multi-organ pharmacokinetic modelling in vitro.
Study Design
Experimental Design
Establish organ modules independently
NANOSTACKS™ Liver MPS (hepatocyte triculture) and Brain MPS (neuronal co-culture) each assembled and matured separately in 24-well plates for 14 days.
Connect organ systems
Medium conditioned by the liver compartment transferred to the brain compartment at defined intervals (every 24–48 h) to simulate systemic circulation and metabolite exposure.
Compound exposure
Test compounds applied to the liver compartment (apical). Metabolite profiles characterised in transfer medium by LC-MS/MS. Metabolite-conditioned medium then applied to the brain compartment.
Parallel and combined readouts
Hepatotoxicity, liver function, and metabolic activity measured in the liver module. Neurotoxicity, network activity, and inflammatory responses measured in the brain module, with and without liver conditioning.
Model Configuration
Model Configuration and Cell Types
Liver Module
- Primary human hepatocytes (PHH) — basal layer
- Liver sinusoidal endothelial cells (LSECs) — apical
- Kupffer cells — apical (immune component)
- Pore size: 1.0 µm (hepatocyte layer)
Brain Module
- Neurons (SH-SY5Y or iPSC-derived) — basal layer
- Astrocytes — intermediate layer
- Brain microvascular endothelial cells — apical
- MEA-compatible format for functional readouts
For standalone module details, see Liver MPS and Brain MPS product pages.
Readouts & Methods
Readouts and Methods
Liver module — function & toxicity
- Albumin secretion and urea synthesis
- ALT / AST hepatocellular injury markers
- LDH cytotoxicity
- CYP3A4 / CYP1A2 activity (metabolic competence)
Metabolite characterisation
- LC-MS/MS metabolite profiling of transfer medium
- Reactive metabolite trapping assays
- Metabolite identity comparison (parent vs liver-conditioned)
Brain module — neurotoxicity
- MEA spontaneous network activity (MFR, burst frequency)
- Neurite integrity imaging (β-III tubulin)
- LDH neurotoxicity from liver-conditioned medium
- Calcium imaging (network excitability)
Systemic inflammatory signalling
- Cytokine panel in transfer medium (IL-6, TNF-α)
- Cross-organ inflammatory signalling assessment
- Compartment-specific cytokine measurements
Key Findings
Key Findings
- 1Liver-conditioned medium transferred to the brain compartment produced measurable changes in neuronal network activity not observed with parent compound alone — demonstrating metabolite-driven neurotoxicity.
- 2Hepatic metabolism generated transformation products detected by LC-MS/MS, some not present in standard reference databases, highlighting the value of human-specific metabolite profiling.
- 3Neurotoxicity thresholds were 2–8x lower for liver-conditioned medium compared to parent compound, consistent with metabolic bioactivation.
- 4Cytokine profiling of transfer medium revealed liver-to-brain inflammatory signalling, with elevated IL-6 correlating with microglial activation markers in the brain module.
- 5Both modules maintained functional stability over the 14-day connected culture period, validating the feasibility of extended multi-organ studies.
Significance
Why It Matters
Single-organ models cannot account for the systemic pharmacokinetic processes that determine actual drug exposure at secondary target organs. The liver-brain axis is particularly relevant because first-pass hepatic metabolism substantially alters compound structure before systemic distribution; some hepatotoxins generate neurotoxic metabolites; and inflammatory mediators released from a drug-injured liver can trigger neuroinflammation and CNS dysfunction.
This approach supports IVIVE and PBPK parameterisation using human-relevant metabolite data, aligned with FDA Modernization Act 2.0 expectations. See Multi-Organ MPS and the FAQ for multi-organ pharmacokinetics discussion.
Platform
Platform Used
This study uses NANOSTACKS™ — Revivocell's modular microphysiological systems platform — with two independent organ modules connected via medium transfer to simulate systemic circulation. Each module operates in a standard 24-well plate format under orbital shaking. No external pumps or specialist equipment are required. See What is MPS? for platform context.
Data & Figures
Experimental Data & Figures
Figures included in the full application note:
- Fig. 1A — HepaRG® cell viability over 9 days on NANOSTACKS™ (RLU, mean ± SEM)
- Fig. 1B — CYP3A4 activity over 9 days (RLU)
- Fig. 1C — CYP3A4 activity normalised to viability; no significant difference across timepoints
- Fig. 1D — Widefield image of HepaRG® cells on NANOSTACKS™ day 1 (10×, scale bar 250 µm)
- Fig. 2A — Widefield images of triculture (HepaRG®, U-138 MG, SH-SY5Y) on day 3
- Fig. 2B — Triculture scheme (NANOSTACKS™ layer arrangement)
- Fig. 2C — Paclitaxel dose-response curve: triculture vs. monoculture IC50 values
View all figures, microscopy images, and dose-response curves in the embedded PDF below.
Related Resources
Related Resources
Full Application Note