Skip to content
← Back to browse
LIVER Publication-derived

Construction of a metastasis model for liver cancer spheroids to hepatobiliary organoids facilitated by holographic acoustic tweezers

Source Li et al., 2025 · Institute of Scientific Instrumentation, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences; Harbin Medical University · 10.1016/j.xcrp.2025.102604

👤 Jialong Li, Dingyuan Liu, Zeping Gao, Mengting Sun, Shuo Wang, Lihong Zhang, Ye Yang, Hongyu Wang, Hairong Zheng, Ye Tian, Teng Ma ⏱ 42 days 📋 15 phases 🧫 Human iPSC (hiPSC); Human liver cancer cell line (HuH-7 expressing GFP)

Abstract

This protocol describes the construction of an in vitro liver cancer metastasis model using holographic acoustic tweezers to assemble hepatobiliary organoids (HBOs) derived from human iPSCs with liver cancer spheroids (HuH-7 cells). The protocol includes differentiation of hiPSCs into HBOs, generation of uniform GFP-expressing HuH-7 spheroids, validation of acoustic field safety, and assembly of unifocal and multifocal metastasis models to recapitulate cancer infiltration into normal or ethanol-treated liver tissue.

Cell source
Human iPSC (hiPSC); Human liver cancer cell line (HuH-7 expressing GFP)
Application
Disease modeling; Cancer metastasis research and drug screening

Protocol overview

80 steps across 15 phases

hiPSC Maintenance and Preparation Day 0
  1. 1 Culture hiPSCs on coated plates
  2. 2 Prepare HuH-7 cells for spheroid formation
HuH-7 Spheroid Formation Day 0–4
  1. 1 Seed HuH-7 cells into ultra-low attachment plates
  2. 2 Culture spheroids for 4 days with medium changes
  3. 3 Verify spheroid uniformity and morphology
Hepatobiliary Organoid Differentiation from hiPSCs Day 0–35
  1. 1 Dissociate and seed hiPSCs for differentiation
  2. 2 Day 1–2: Reduce Y-27632 concentration
  3. 3 Day 4: Transition to hepatic mesoderm induction
  4. 4 Day 9: Embed organoids in Matrigel and expand medium
  5. 5 Day 15: Transition to hepatocyte maturation medium
  6. 6 Day 25–35: Final maturation with dexamethasone, vitamin K2, and lithocholic acid
Safety Validation of 3D Acoustic Fields on HBOs and Spheroids During phase 3 (optional parallel assessment)
  1. 1 Expose samples to acoustic fields at 24 V, 2% duty cycle
  2. 2 Perform immunofluorescence staining for PCNA and CASP-3
  3. 3 Perform bulk RNA sequencing analysis
  4. 4 Quantify apoptosis and proliferation rates
Functional Characterization of Hepatobiliary Organoids Day 35
  1. 1 Assay ICG uptake and release (liver metabolic function)
  2. 2 Perform PAS and ORO staining (glycogen and lipid storage)
  3. 3 Measure urea secretion (hepatocyte synthetic function)
  4. 4 Assay alkaline phosphatase (ALP) activity (biliary function)
  5. 5 Perform immunofluorescence for structural markers (ALB and CK19)
Hepatobiliary Organoid Characterization by Western Blot During phase 3 (sampling at days 0, 4, 9, 35)
  1. 1 Collect and lyse organoid samples
  2. 2 Quantify total protein and prepare samples
  3. 3 Perform SDS-PAGE and transfer to PVDF membrane
  4. 4 Block and incubate with primary antibodies
  5. 5 Incubate with secondary antibodies and detect
  6. 6 Quantify protein levels and perform statistical analysis
Holographic Acoustic Field Design and Validation Prior to organoid-spheroid assembly
  1. 1 Simulate acoustic fields using COMSOL Multiphysics
  2. 2 Fabricate and characterize 2D ultrasound transducer array
  3. 3 Generate holographic acoustic fields using iterative backpropagation algorithm
  4. 4 Measure acoustic field pressure distribution using hydrophone
  5. 5 Compare simulation with experimental measurements
Construction of Organoid-Spheroid Online Assembly Platform (OSOAP) Prior to assembly
  1. 1 Assemble acoustic tweezers system and cell culture chamber
  2. 2 Validate system assembly and acoustic transmission
  3. 3 Perform sterility testing and calibration
Construction of Unifocal Liver Cancer Metastasis Model Day 35 (organoids) + Day 4 (spheroids)
  1. 1 Prepare HBOs and HuH-7 spheroids for assembly
  2. 2 Position HBO and spheroid within acoustic field region
  3. 3 Activate 1.6λ Vortex acoustic field to move spheroid toward HBO
  4. 4 Switch to 1.6λ asymmetric Vortex to place spheroid on HBO surface
  5. 5 Increase Vortex aperture and maintain contact for 15 min
  6. 6 Remove acoustic field and initiate culture in OSOAP
  7. 7 Maintain culture for 18 h and perform end-point characterization
Construction of Multifocal Liver Cancer Metastasis Model Day 35 (organoids) + Day 4 (spheroids)
  1. 1 Prepare HBO and three HuH-7 spheroids for assembly
  2. 2 Sequentially position each spheroid and move toward HBO
  3. 3 Stabilize first spheroid with larger Vortex aperture
  4. 4 Switch to second spheroid and repeat movement and stabilization
  5. 5 Repeat for third spheroid
  6. 6 Remove acoustic fields and initiate multifocal assembloid culture
  7. 7 Maintain culture for 18 h and characterize
Characterization of Liver Cancer Metastasis Models (Unifocal and Multifocal) After 18 h culture in OSOAP
  1. 1 Fix assembloids and remove Matrigel residue
  2. 2 Perform immunofluorescence for ALB and CK19 (structural integrity)
  3. 3 Perform immunofluorescence for CASP-3 (viability assessment)
  4. 4 Quantify infiltration distance and area
  5. 5 Perform statistical comparison between unifocal and multifocal models
  6. 6 Perform bulk RNA sequencing on model and control assembloids
  7. 7 Create heatmap of cancer metastasis-related genes
  8. 8 Compare model genes with animal model datasets (GEO database)
Construction of Alcoholic Liver Disease (ALD)-Modeled HBOs Day 35 onwards
  1. 1 Prepare EtOH treatment medium
  2. 2 Treat HBOs with 100 mM EtOH for 7 days
  3. 3 Monitor morphological changes during EtOH treatment
Characterization of ALD-Modeled HBOs Day 42 (7 days post-EtOH initiation)
  1. 1 Assess fibrosis by Masson and Sirius Red staining
  2. 2 Quantify fibrosis and perform statistical analysis
  3. 3 Assess steatosis (lipid accumulation) by Nile Red staining
  4. 4 Quantify lipid content
  5. 5 Assess oxidative stress via ROS assay
  6. 6 Quantify ROS levels
  7. 7 Measure pro-collagen type I secretion
  8. 8 Measure pro-inflammatory cytokines (IL-6, TNF-α)
  9. 9 Perform bulk RNA sequencing on ALD-modeled HBOs
  10. 10 Create heatmaps of ALD-related genes
Construction of Metastasis Models Using EtOH-Treated HBOs After ALD characterization (day 42+)
  1. 1 Prepare EtOH-treated HBO and HuH-7 spheroids for assembly
  2. 2 Construct unifocal metastasis model: single spheroid + EtOH-treated HBO
  3. 3 Remove acoustic field and culture unifocal assembloid for 18 h
  4. 4 Construct multifocal metastasis model: three spheroids + EtOH-treated HBO
Characterization of Metastasis Models with EtOH-Treated HBOs and Comparison with Normal HBOs After 18 h culture in OSOAP
  1. 1 Fix assembloids and perform immunofluorescence
  2. 2 Quantify infiltration distance and area for EtOH-treated models
  3. 3 Compare infiltration between normal and EtOH-treated HBOs
  4. 4 Quantify apoptosis in EtOH-treated vs. normal assembloids
  5. 5 Record and analyze time-lapse video of infiltration dynamics
  6. 6 Perform bulk RNA sequencing on EtOH-treated metastasis models
  7. 7 Create comparative heatmaps: normal vs. EtOH-treated metastasis signatures

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Li et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence