Primary human hepatocytes in spheroid formation to study hepatitis C infection.
BACKGROUND
Hepatitis C (HCV) is a worldwide health problem, affecting nearly 170 million people. Current models for studying Hepatitis C have focused primarily on the use of poorly permissive cell lines and viral constructs, because of the lack of a suitable animal model or an in vitro system for studying functional infection. As hepatocytes are the primary reservoir for the virus in vivo, we report on a model using primary human hepatocytes cultured in spheroid formation.


METHODS
The hepatocytes were harvested from uninfected liver resections and cultured as spheroids (that promotes a differentiated phenotype) or monolayers. Spheroids expressed the putative receptors CD81 and human scavenger receptor B1 in a variable pattern throughout the culture period. Samples were inoculated with infectious HCV serum, and HCV RNA was detected using RT-PCR. RNA was detected in the cells and culture medium by 3 days and 5 days after inoculation, respectively. Selection of HVR1 variants occurred in a differential pattern based on culture technique, suggesting that viral selection was dependent on host phenotype. Detection of NS5A by Western blot analysis of infected samples and immunofluorescence for HCV core protein was seen only in infected spheroids.


CONCLUSIONS
The use of spheroid formation to study Hepatitis C is associated with the establishment of HVR1 selection and functional infection. This represents a promising alternative model to study Hepatitis C.
Chong
Tae W
TW
Department of Surgery, Surgical Infectious Disease Laboratory, University of Virginia, Charlottesville, Virginia 22908, USA. twc2u@virginia.edu
Smith
Robert L
RL
Hughes
Michael G
MG
Camden
Jeremy
J
Rudy
Christine K
CK
Evans
Heather L
HL
Sawyer
Robert G
RG
Pruett
Timothy L
TL
D015703:Antigens, CD / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D045744:Cell Line, Tumor; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C; D018197:Hepatoblastoma; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism / Q000821:virology*; D006801:Humans; D008113:Liver Neoplasms; D008854:Microscopy, Electron; D018807:Polymorphism, Single-Stranded Conformational; D012367:RNA, Viral / Q000032:analysis; D051116:Receptors, Scavenger / Q000378:metabolism; D018874:Spheroids, Cellular / Q000648:ultrastructure / Q000821:virology*; D060225:Tetraspanin 28
D015703:Antigens, CD; C554798:CD81 protein, human; D012367:RNA, Viral; D051116:Receptors, Scavenger; D060225:Tetraspanin 28