HBV viral replication in extrahepatic plays an important role in the pathogenesis of extrahepatic syndromes. Currently, cell culture models for starting extrahepatic HBV infection are limited. We present an in vitro non-hepatic infection model to help identify novel host factors which affect HBV replication and investigate HBV-related kidney diseases.
Compared to traditional HBV infection models, we adopted and engineered 293T cell line, 293T-NE-3NR, and co-cultured it with HepG2.2.15. HBV infection should be performed in a biosafety level two or biosafety level three laboratory. Laboratory safety practices should be followed to ensure the safety of laboratory personnel and all researchers should be vaccinated and detect HBS antibody positive before performing HBV experiments.
Begin by culturing the HepG2.2.15 cells keeping in mind that the cell supernatant as well as all the tips, flasks, plates, and tubes that come in contact with HepG2.2.15 should be soaked in 2%virucide overnight prior to disposal. Remove the vial containing HepG2.2.15 cells from liquid nitrogen and thaw it in gentle swirling in a 37 degree Celsius water bath. Transfer the cells to a 25 centimeter square tissue culture flask and add four milliliters of complete culture medium.
Culture the HepG2.2.15 cells at 37 degrees Celsius and 5%carbon dioxide in a humidified incubator. Change the medium every three days. When ready, collect the cell supernatant in a 50 milliliter centrifuge tube.
Close the lid firmly and wrap it with paraffin film. To remove the cell fragments, filter the HepG2.2.15 supernatant with a 0.45 micrometer membrane into a new 50 milliliter centrifuge tube. Then add 14 milliliters of filtered supernatant to a virus concentrator column and close the lid.
Centrifuge the column at 3, 200 times g for 35 minutes in a horizontal spinning centrifuge and collect the HepG2.2.15 supernatant concentrate into a 1.5 milliliter tube. Run real-time PCR to ensure that the concentration if HBV DNA in the HepG2.2.15 supernatant concentrate is within the standard curve range. Dilute the concentrate 20-fold by adding 10 microliters to 190 microliters of DMEM in a 1.5 milliliter microcentrifuge tube.
Along with the supernatant concentrate, prepare a negative control, positive control, and four dilutions of the quantitative reference. Add 450 microliters of HBV DNA extraction buffer to each sample and centrifuge them at 12, 000 times g for five minutes at room temperature. Combine 27 microliters of PCR master mix and three microliters of Taq polymerase enzyme per sample in PCR tubes placed on ice.
Then add 20 microliters of the centrifuged sample to each tube. Perform real-time PCR according to manuscript directions and export the CT values to obtain a standard curve. Divide the HepG2.2.15 supernatant concentrate into aliquots and store it at minus 80 degrees Celsius until ready to use.
Prepare infection medium according to manuscript directions. Then seed HepG2-NE in two wells, 293T-NE-3NR cells in two wells, and 293T-NE in one well. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide in a humidified incubator for 24 hours.
After the incubation, observe the cells and proceed with infection if they're healthy. Add 500 microliters of the infection complex to each well and incubate the cells for another 24 hours. Wash the cells twice with 500 microliters of PBS.
Then add one milliliter of medium to each well. Change the medium gently every two days. On day 11, gently wash the cells twice with 500 microliters of PBS and fix the cells with ice cold methanol for immunofluorescence.
Seed 100, 000 cells per well of HepG-NE into two wells, 293T-NE-3NRs into two wells, and 293T-NE into one well of the plate. Seed 100, 000 cells per well of HepG2.2.15 into five membrane inserts in a separate six-well plate. Incubate the cells for 24 hours.
After the incubation, ensure that the cells are all adherent and in good state. Discard the medium in the six-well plate and the cell membrane inserts. Then place the membrane inserts with HepG2.2.15 into the six-well plate seeded with HepG-NE, 293T-NE-3NRs, and 293T-NE.
Incubate the cells at 37 degrees Celsius and 5%carbon dioxide in a humidified incubator, changing the medium every three to four days. After 10 days, remove the membrane inserts, gently wash the cells with PBS twice, and fix the cells with ice cold methanol for immunofluorescence. Incubation with the HBcAg primary antibody and DAPI resulted in a distinct staining when observed with a 10X objective on a fluorescent inverted microscope.
Nuclear localization was confirmed by staining with DAPI and NTCP-EGFP expression was identified with green fluorescence. The expression sites of HBcAg were located with red fluorescence. When DAPI, NTCP and HBcAg are in the same cell, the cell has been successfully infected with HBV.
The Cyclosporin A group was the negative control because it prevents HBV from entering cells by blocking NTCP. HBcAg was detected in 293T-NE-3NRs, but not in 293T-NE cells indicating NTCP is not the only factor essential for HBV infection in 293T. Good cell status and efficient operation are the key points to getting successful infection results.
Gentle washing and changing the media after infection is also important. As an alternative, HBV infection method with hepatoma-based HepG2-NE cells has higher infection efficiency than this method and is suitable for anti-HBV drug screening. Modeling HBV infection in 293T-NE-3NR is a useful compliment to the traditional cell model due to the non-hepatic background of these cells.
This method will facilitate the study of HBV infection in non-hepatic cells as well as host factors required for liver of HBV.
