How advanced 3D cell culture technology is transforming our understanding of hepatitis B virus and accelerating the search for a cure
For decades, hepatitis B virus (HBV) researchers have faced a persistent challenge: how to study this complex pathogen in laboratory conditions that truly mimic the human liver. With approximately 254 million people living with chronic HBV infection globally, the lack of effective laboratory models has significantly hampered progress toward a cure 7 .
The fundamental limitation of traditional 2D cell culture lies in its inability to recapitulate the intricate architecture of human liver tissue. In our bodies, hepatocytes exist in a complex 3D environment, surrounded by other cell types and extracellular matrix, communicating with neighbors in all directions 8 .
3D spheroids exhibit enhanced liver-specific functions compared to 2D counterparts, with improved expression of metabolic enzymes and better albumin production 3 .
3D liver cultures demonstrate higher sensitivity for detecting hepatotoxic chemicals compared to traditional 2D systems 3 .
The 3D environment better supports HBV infection as the virus primarily infects fully functional, mature liver cells 9 .
The newly developed 3D spheroid system represents a significant advancement in HBV research methodology. At its core are mouse-passaged primary human hepatocytes (mpPHHs)—a specialized type of liver cell that has been "rejuvenated" through a process of engrafting human hepatocytes into specially engineered mice 2 .
To validate their novel system, the research team conducted a crucial experiment demonstrating both the longevity of their 3D spheroids and their practical application in drug testing 1 2 .
Mouse-passaged primary human hepatocytes were initially plated in conventional 2D format 2 .
Cells were washed and shipped overnight at room temperature—demonstrating practical robustness 2 .
Cells were transferred to ultra-low attachment plates to facilitate spontaneous spheroid formation 2 3 .
Spheroids were infected with HBV to establish the de novo infection model 2 .
Establishing a successful 3D spheroid system for HBV research requires several key components, each playing a critical role in the process.
These "rejuvenated" hepatocytes, prepared by engrafting PHHs into FNRG mice, show enhanced infectability and avoid rapid dedifferentiation 2 .
HCM medium supplemented with 2% DMSO, gentamicin, and ciprofloxacin maintains hepatocyte function and prevents contamination 2 .
Essential for HBV entry into hepatocytes, though mpPHHs naturally express sufficient levels without artificial introduction 7 .
| Reagent/Equipment | Function | Alternative/Note |
|---|---|---|
| mpPHHs | Primary functional hepatocytes with enhanced longevity | Traditional PHHs can be used but with limitations |
| ULA Plates | Facilitates spontaneous spheroid formation | Hanging drop plates are an alternative method |
| HCM Medium | Supports hepatocyte function and viability | Must be supplemented with DMSO for optimal function |
| HBV Capsid Assembly Modulators | Experimental therapeutics for validation studies | Representative of antiviral drug classes |
| Detection Assays | Measure HBV DNA, RNA, antigens, and hepatocyte function | Include ELISA, qPCR, immunofluorescence |
The development of this robust 3D spheroid system marks a significant milestone in hepatitis B research. By bridging the gap between conventional cell culture and the complex human liver environment, this technology enables scientists to study the complete HBV life cycle—including the persistent cccDNA responsible for viral rebound after treatment cessation 2 7 .
This advancement comes at a critical time in the global effort to eliminate viral hepatitis. The World Health Organization has set an ambitious target to eliminate viral hepatitis as a public health threat by 2030, a goal that requires accelerated development of effective treatments 7 .
The 3D spheroid system provides a much-needed platform for high-throughput screening of novel antiviral compounds and combination therapies.
Future refinements may incorporate additional cell types like immune cells to create even more sophisticated simulated liver environments.
The era of 3D modeling has truly opened new dimensions in hepatitis B research, offering hope for the millions affected by this global health challenge.
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