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LIVER Publication-derived

3D Hepatospheres in PEG-Alginate-Gelatin Cryogel: In Vitro and In Vivo Hepatitis C Virus Model

Source Jayal et al. · Indian Institute of Technology Kanpur, Department of Biological Sciences and Bioengineering · 10.1002/bit.27651

👤 Priyanka Jayal, Padmanava Behera, Ranajoy Mullick, Subbaraya G. Ramachandra, Saumitra Das, Ashok Kumar, Anjali Karande ⏱ 32 days 📋 10 phases 🧫 Human hepatocellular carcinoma cells (Huh7.5)

Abstract

This protocol describes the generation and characterization of functionally robust 3D spheroids of HCV-permissive Huh7.5 hepatoma cells supported on a PEG-alginate-gelatin (PAG) cryogel scaffold. The 3D culture exhibits elevated expression of hepatic markers, HCV co-receptors, and drug-metabolizing enzymes compared to 2D culture, and supports robust HCV infection and replication in vitro. Implanted 3D constructs establish as ectopic human hepatic tissue in athymic nude mice with sustained viability up to 32 days, providing a physiologically relevant platform for HCV pathogenesis studies and antiviral drug screening.

Cell source
Human hepatocellular carcinoma cells (Huh7.5)
Application
Disease modeling and drug screening for Hepatitis C virus infection

Protocol overview

81 steps across 10 phases

PAG Cryogel Synthesis and Characterization Pre-culture
  1. 1 Prepare alginate solution
  2. 2 Mix cryogel components
  3. 3 Pour and freeze cryogel
  4. 4 Thaw and wash cryogel
  5. 5 Lyophilize cryogel
  6. 6 Sterilize cryogel discs
Smart Polymer Preparation and Hepatospheroíd Formation Pre-culture and Day 0–2
  1. 1 Prepare PNIPAAm stock solution
  2. 2 Prepare Eudragit S-100 stock solution
  3. 3 Set up spinner culture for hepatosphere formation
  4. 4 Harvest hepatospheres
3D Cryogel-Based Hepatospheroíd Culture Day 0–30
  1. 1 Prepare PAG cryogel discs
  2. 2 Seed hepatospheres on cryogel
  3. 3 Collect culture supernatant for HSA quantification
  4. 4 Assess cell viability by MTT assay
  5. 5 Replace culture medium
Histological and Immunofluorescence Characterization Day 6–18
  1. 1 Fix 3D culture for scanning electron microscopy
  2. 2 Dehydrate and prepare SEM samples
  3. 3 Image by scanning electron microscopy
  4. 4 Fix 3D culture for confocal microscopy
  5. 5 Dehydrate and paraffin-embed samples
  6. 6 Section paraffin-embedded samples
  7. 7 Deparaffinize and rehydrate sections
  8. 8 Perform antigen retrieval
  9. 9 Block and permeabilize sections
  10. 10 Incubate with primary antibodies
  11. 11 Wash and detect primary antibodies
  12. 12 Counterstain nuclei and mount
  13. 13 Image by confocal microscopy
Gene Expression and Molecular Analysis Day 6–12
  1. 1 Extract total RNA
  2. 2 Synthesize cDNA
  3. 3 Perform real-time qPCR
  4. 4 Extract proteins for Western blot analysis
  5. 5 Quantify protein by Bradford assay
  6. 6 Prepare protein samples for SDS-PAGE
  7. 7 Perform SDS-PAGE electrophoresis
  8. 8 Transfer proteins to nitrocellulose membrane
  9. 9 Block membrane and incubate with primary antibody
  10. 10 Wash and incubate with secondary antibody
  11. 11 Develop and visualize blot
HCV Binding and Infection Studies Day 12–24
  1. 1 Prepare HCV-like particles (HCV-LPs) genotype-3a
  2. 2 Label HCV-LPs for 2D binding studies
  3. 3 Perform binding kinetics on 2D Huh7.5 cells
  4. 4 Image 2D binding samples
  5. 5 Perform binding kinetics on 3D culture
  6. 6 Process and stain 3D binding samples for immunofluorescence
  7. 7 Detect bound HCV-LPs in 3D culture
  8. 8 Counterstain, mount, and image 3D binding samples
JFH1 HCV Infection and Replication Day 12–18
  1. 1 Prepare pJFH1 construct and generate viral RNA
  2. 2 Transfect Huh7.5 cells with HCV RNA
  3. 3 Harvest and quantify infectious HCV
  4. 4 Infect 3D and 2D cultures with HCV
Viral RNA Quantification and Assessment of Replication Day 15–18
  1. 1 Extract total RNA from infected 3D constructs
  2. 2 Synthesize cDNA from HCV RNA (negative strand)
  3. 3 Quantify viral RNA by real-time RT-PCR
  4. 4 Quantify positive-strand viral RNA (released virus)
  5. 5 Quantify miRNA-122 transcript levels
  6. 6 Assess HSA expression changes upon HCV infection
  7. 7 Detect HCV envelope proteins in infected tissue sections
  8. 8 Visualize HCV-infected cells by confocal microscopy
Antiviral Therapeutic Screening Day 15–18
  1. 1 Select neutralizing monoclonal antibodies
  2. 2 Pre-incubate HCV with neutralizing antibodies
  3. 3 Infect 3D culture with antibody-treated virus
  4. 4 Assess viral replication inhibition
  5. 5 Document therapeutic efficacy
In Vivo Implantation in Athymic Nude Mice Pre-implantation (day 6 in vitro) to 32 days post-implantation
  1. 1 Prepare 3D constructs for implantation
  2. 2 House and prepare experimental animals
  3. 3 Anesthetize mice and prepare surgical site
  4. 4 Perform surgical implantation
  5. 5 Post-operative pain management
  6. 6 Collect blood samples at specified time points
  7. 7 Quantify human serum albumin (HSA) in mouse serum
  8. 8 Assess liver and kidney function
  9. 9 Euthanize mice and necropsy
  10. 10 Process excised implants and organs for histopathology
  11. 11 Prepare histological sections
  12. 12 Perform H&E staining
  13. 13 Assess tissue integrity and vascularization
  14. 14 Perform immunofluorescence on excised implants
  15. 15 Detect HSA in implant sections
  16. 16 Counterstain and visualize implant sections
  17. 17 Culture excised implants ex vivo

Full SOP

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Attribution

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

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