Dialysis cassette

Cryopreserved plasma samples from de-identified, chronic HBV carriers were kindly provided by Susan Stramer (American Red Cross, Gaithersburg, MD). To purify infectious HBV virions and remove coagulation factors the patient plasma samples were loaded onto a 1 ml HiTrap heparin column (GE, Fairfield, CA). The samples once loaded were then washed with 2-column volumes worth of wash buffer (20 mM phosphate buffer, 50 mM NaCl, pH = ~ 7). This helped aid in the removal of non-infectious sub-viral particles as well as remove some of the coagulation factors present in the patient plasma. The infectious virus was then eluted using elution buffer (20 mM phosphate buffer, 2 M NaCl, pH = ~ 7). Once the purified infectious HBV was isolated it was dialyzed using a 3 ml dialysis cassette (Millipore, Darmstadt Germany) in sterile 1× PBS. After dialysis, virus was stored at 4 °C until use 24 h later.

HepG2.2.15 cells^{7 (link)} were grown in media containing tetracycline until they reached a confluency of 100%. At this time media was changed to DMEM F12 media supplemented with 10% FBS, 1% Pen/Strep. Media from the HepG2.2.15 culture was collected every 2 to 3 days for approximately 4 weeks. The collected media was sterile filtered through a 0.22 μm filter (Millipore, Darmstadt, Germany) and was then concentrated ca. 100-fold using a stir cell concentrator (Millipore, Darmstadt, Germany). After concentration, the virus was run over a 5 mL HiTrap heparin column (GE, Fairfield, CA) to further concentrate and purify infectious virus particles from non-infectious sub-viral particles. Concentrated virus was applied to a heparin column, which was washed with 5-column volumes of wash buffer (20 mM phosphate buffer, 50 mM NaCl, pH = ~ 7). Afterwards, the virus was eluted with elution buffer (20 mM phosphate buffer, 2 M NaCl, pH = ~ 7). Once all virus was eluted, the viral stock was dialyzed using a dialysis cassette (Millipore, Darmstadt, Germany). After dialysis, virus was aliquoted into cryovial tubes and cryopreserved at −80 °C until use.