中文导读
肝脏是药物代谢的主要场所。本文介绍肝脏芯片的构建、肝细胞长期培养及其在 ADMET 评价中的应用。
以下为英文全文（English full text）
1. Introduction: The Liver as a Central Hub in Drug Development
The liver is the primary site of drug metabolism, responsible for the biotransformation of xenobiotics through Phase I (oxidation, reduction, hydrolysis) and Phase II (conjugation) reactions. Approximately 75% of marketed drugs undergo hepatic metabolism, and drug-induced liver injury (DILI) accounts for over 50% of acute liver failure cases in the United States and remains the leading cause of drug attrition during clinical development and post-market withdrawal [1]. The urgent need for predictive human liver models has driven intensive development of liver-on-chip (LoC) technology, which recapitulates the hepatic microarchitecture, hemodynamic environment, and multicellular interactions essential for maintaining drug-metabolizing enzyme function [2].
Unlike conventional 2D hepatocyte monolayers, which rapidly lose CYP450 enzyme activity and polarity within 48–72 hours, microfluidic liver chips sustain hepatocyte function for weeks through continuous perfusion, co-culture with non-parenchymal cells, and recreation of physiological shear stresses. These platforms have demonstrated improved prediction of metabolic clearance, drug-drug interactions, idiosyncratic toxicity, and cholestatic mechanisms compared to both static cultures and animal models [3].
2. Hepatic Microarchitecture and Physiological Parameters
The liver's functional unit is the hepatic lobule, a hexagonal structure approximately 1–2 mm in diameter organized around a central vein. Blood flows from the portal triad (portal vein, hepatic artery, bile duct) through sinusoidal capillaries lined with fenestrated liver sinusoidal endothelial cells (LSECs), past hepatocyte plates separated by the Space of Disse, and drains into the central vein. This architecture creates oxygen and metabolic zonation: periportal (Zone 1) hepatocytes are exposed to high oxygen (~60–70 mmHg) and express high levels of gluconeogenic enzymes, while pericentral (Zone 3) hepatocytes experience lower oxygen (~30–35 mmHg) and specialize in glycolysis, lipogenesis, and CYP2E1/CYP3A4-mediated drug metabolism [4].
Critical physiological parameters for LoC design include:
- Sinusoidal blood flow velocity: 100–500 μm/s
- Wall shear stress in sinusoids: 0.2–0.6 dyn/cm²
- Oxygen gradient across lobule: 60–70 mmHg (portal) to 30–35 mmHg (central)
- Hepatocyte width: 20–30 μm
- Sinusoid height: 5–10 μm
- Space of Disse width: 0.5–1.0 μm
- Bile canaliculus diameter: 1–2 μm
- Albumin production rate: 10–15 g/day (whole liver)
- Urea synthesis rate: 20–30 g/day (whole liver)
3. Liver-on-Chip Design Principles and Construction
3.1 Membrane-Based Bilayer Devices
The most widely adopted LoC architecture employs two parallel microfluidic channels separated by a porous membrane. Hepatocytes are cultured on one side of the membrane (apical/basolateral orientation), while endothelial cells line the opposite channel, recreating the sinusoidal interface. The CN Bio Innovations PhysioMimix platform and similar commercial systems use this configuration with a microporous polyester membrane (pore size 0.4–3.0 μm, pore density 1–4×10⁶ pores/cm²) to enable molecular exchange while maintaining cellular compartmentalization [5].
Typical channel dimensions are 200–1000 μm in width, 100–250 μm in height, and 10–30 mm in length. Perfusion flow rates of 5–50 μL/min generate wall shear stresses of 0.1–1.0 dyn/cm², which promotes hepatocyte polarization without inducing detachment. The membrane surface is coated with collagen I (50–100 μg/mL), fibronectin (10–20 μg/mL), or Matrigel to facilitate cell adhesion and bile canaliculus formation [5].
3.2 Sinusoid-Mimicking Microchannel Designs
Advanced LoC devices recreate the hepatic sinusoid geometry using single or dual microchannels with integrated microposts or micropatterned grooves that guide hepatocyte and LSEC alignment. Mi et al. (2018) developed a liver sinusoid chip based on laminar flow patterning and endothelial cell self-assembly, achieving physiologically relevant albumin synthesis and urea production rates [6]. The device featured a central channel (400 μm wide, 100 μm high) with adjacent perfusion channels for artificial blood and bile flow, enabling studies of bile acid transport and cholestatic drug mechanisms [6].
3.3 3D Spheroid and Microtissue Integration
Hepatocyte spheroids (100–300 μm diameter) formed in hanging-drop or ultra-low attachment plates can be transferred into microfluidic chips for perfusion culture. This approach combines the enhanced cell-cell contact and polarity of 3D spheroids with the dynamic nutrient delivery of microfluidics. Theobald et al. (2019) demonstrated a multi-compartment liver-kidney organ-on-chip integrating hepatocyte spheroids with renal proximal tubule cells, enabling sequential metabolic activation and renal clearance of vitamin D3 [7].
3.4 Zonation-Recreating Gradient Devices
To replicate the oxygen and metabolic zonation of the hepatic lobule, gradient-generator LoC devices create stable oxygen gradients across hepatocyte cultures. Christmas-tree microfluidic mixers deliver media with varying oxygen tensions to parallel culture chambers, while gas-permeable PDMS walls enable oxygen diffusion. These devices allow simultaneous study of zone-specific drug metabolism and toxicity, addressing the zonal selectivity of drugs such as acetaminophen (pericentral toxicity) and allyl alcohol (periportal toxicity) [4].
4. Cell Culture Protocols and Co-Culture Systems
Primary Human Hepatocytes (PHH)
PHH remain the gold standard for LoC applications due to their high CYP450 activity and transporter function. Seeding densities of 1–5×10⁶ cells/cm² are typically used. Cells are seeded in hepatocyte attachment medium (e.g., Williams' E medium with 10% FBS, 1% penicillin-streptomycin, 4 μg/mL insulin, 1 μM dexamethasone) and allowed to attach for 4–6 hours before initiating perfusion. Media is switched to serum-free maintenance medium within 24 hours to promote polarization [2].
Hepatocyte Co-Culture
Hepatocyte mono-cultures rapidly decline in function, but co-culture with non-parenchymal cells (NPCs) extends viability and metabolic activity. Standard co-culture configurations include:
- Hepatocytes + LSECs: LSECs are seeded on the opposite side of the porous membrane or adjacent to hepatocytes in shared channels. This arrangement supports albumin and urea production for >4 weeks.
- Hepatocytes + 3T3-J2 Fibroblasts: Fibroblasts provide paracrine signals that maintain hepatocyte differentiation. Khetani and Bhatia's micropatterned co-culture system (μPCC) demonstrated stable CYP450 activity for >6 weeks [8].
- Hepatocytes + Kupffer Cells: Inclusion of macrophages enables modeling of immune-mediated hepatotoxicity, including inflammatory cytokine release and idiosyncratic DILI triggered by lipopolysaccharide (LPS) priming.
- Hepatocytes + Stellate Cells: Hepatic stellate cell activation and collagen deposition can be modeled, enabling studies of fibrogenic drug effects [2].
iPSC-Derived Hepatocytes
iPSC-hepatocytes offer renewable, genetically defined cell sources for LoC applications. When cultured in liver chips, iPSC-hepatocytes show enhanced maturation compared to 2D culture, with improved albumin secretion, CYP3A4 activity, and bile acid transport. However, CYP450 expression levels remain approximately 30–50% of those in primary hepatocytes, and continued protocol optimization is needed for full metabolic equivalence [9].
5. Functional Validation and Analytical Readouts
Metabolic Function Markers
- Albumin secretion (ELISA): 50–200 μg/mL/day in healthy PHH chips
- Urea synthesis (colorimetric assay): 200–800 μg/mL/day
- CYP450 activity: Measured using luminogenic or fluorogenic substrates for CYP1A2, CYP2B6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4
- Phase II conjugation: UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) activities using 7-hydroxycoumarin or p-nitrophenol substrates
- Bile acid transport: Biliary excretion of cholyl-lysyl-fluorescein (CLF) or taurocholate
Cytotoxicity and Injury Assessment
- Lactate dehydrogenase (LDH) release
- ATP content (CellTiter-Glo)
- Live/dead staining (calcein-AM/ethidium homodimer)
- Caspase-3/7 activation for apoptosis
- Reactive oxygen species (ROS) detection (DCFH-DA)
- Mitochondrial membrane potential (JC-1, TMRM)
- Glutathione (GSH) depletion
Morphological and Molecular Characterization
- Immunofluorescence for tight junction proteins (ZO-1, occludin), polarity markers (MRP2, BSEP), and hepatic markers (albumin, HNF4α)
- Trans-epithelial electrical resistance (TEER) for barrier integrity
- Transmission electron microscopy for bile canaliculus formation and tight junction ultrastructure
- RNA-seq and proteomics for transcriptomic and proteomic profiling
6. ADMET Applications of Liver-on-Chip
Metabolic Clearance Prediction
LoC platforms with PHH enable extended incubation of drug candidates (up to 14 days) with repeated sampling, generating time-course concentration data for clearance estimation. Docci et al. (2022) demonstrated that the PhysioMimix liver-on-chip produced intrinsic clearance (CLint) values for metabolically stable drugs that correlated well with human in vivo data, enabling accurate IVIVE (in vitro-in vivo extrapolation) when combined with mathematical modeling of media evaporation and non-specific binding [5].
Drug-Drug Interaction (DDI) Assessment
LoC models support the evaluation of CYP450 induction and inhibition over physiologically relevant timeframes. Co-administration of CYP3A4 inducers (rifampicin) or inhibitors (ketoconazole) with victim drugs enables quantification of AUC fold-changes and prediction of clinical DDI risk. Multi-organ chips connecting liver and heart tissues can further predict organ-specific DDI consequences, such as the bioactivation of prodrugs by hepatic enzymes and subsequent cardiotoxicity of metabolites [10].
Drug-Induced Liver Injury (DILI)
The FDA's landmark acceptance of Emulate's Liver-Chip for DILI prediction in 2024 validated the technology's regulatory utility. The chip demonstrated sensitivity and specificity exceeding 80% for identifying DILI-positive drugs, including compounds that appeared safe in animal studies but caused human toxicity (e.g., troglitazone, ximelagatran). The inclusion of Kupffer cells and LSECs enabled detection of immune-mediated idiosyncratic DILI, a major challenge for conventional in vitro models [4].
Cholestatic and Steatotic Mechanisms
LoC platforms with functional bile canaliculi enable real-time imaging of bile acid accumulation and transport inhibition. Drugs such as cyclosporine A and bosentan, which inhibit BSEP and MRP2 transporters, cause dose-dependent bile acid retention and canalicular dilatation in LoC models. Steatosis-inducing drugs (e.g., amiodarone, tetracycline) can be identified by lipid droplet accumulation visualized with Oil Red O staining or BODIPY fluorescent probes [2].
7. Technical Specifications and Operating Parameters
8. Conclusion
Liver-on-chip technology has emerged as a transformative platform for ADMET applications, offering predictive power for metabolic clearance, DILI risk, drug-drug interactions, and cholestatic/steatotic toxicity. With regulatory acceptance from the FDA and growing adoption across pharmaceutical companies, LoC platforms are poised to become standard tools in preclinical drug development. GBiowit provides state-of-the-art liver-on-chip platforms, hepatocyte-compatible media, matrices, and expert ADMET screening services to support your research and regulatory needs.