Cell lines and cell culture
We used five human hepatoma cell lines form Korean cell line bank (KCLB) and Merck KGaA. Huh7 (KCLB No. 60104), HepG2 (KCLB No. 88065) as HBV negative, PLC/PRF/5 (KCLB No. 28024), HepG2.2.15 (Merck KGaA, No. SCC249), and Hep3B (KCLB No. 88064) expressing HBsAg. The cell lines were cultured in a 37°C, 5% CO2 incubator and Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS, Thermo Scientific Hyclone) and 1xAntibiotic-Antimyotic (Anti-Anti).
Human Hepatocyte Isolation
Human hepatocytes from patients with high titers of HBsAg and HBV DNA in the blood were used. Approximately 50–70 g of liver tissue from a patient infected with hepatitis B virus and positive for HBsAg was obtained. After connecting two or three catheters filled with William's media to a large vessel exposed to a cross-section of the liver tissue, perfusion solution A [Hanks’ balanced salt solution (HBSS) 250 ml + hydroxyethyl piperazine-ethane-sulfonic acid (HEPES) 2.5 ml + antibiotics 2.5 ml + EDTA 250 µl] was perfused through a peristaltic pump at a flow rate of 30 ml per minute per catheter. After approximately 10 min, a perfusion solution B (HBSS 250 ml + HEPES 2.5 ml + antibiotics 2.5 ml +collagenase 0.125 g) was used for perfusion for another 10 min. After removing the catheter from the liver tissue that had turned yellow due to decomposition, the liver tissue was transferred to another bowl, and cold William's medium was poured. A knife was used to obtain the enzymatically degraded tissue cells.
Immunoglobulins
Three immunoglobulins [control human IgG (Sigma, Dorset, UK), conventional plasma-derived hepatitis B immunoglobulin (cHBIG, GC pharma, Yongin, Korea), and recombinant hepatitis B immunoglobulin and its Fab portion (lenvervimab, GC pharma, Yongin, Korea)] were diluted with Dulbecco’s Phosphate Buffered saline (DPBS) or media corresponding to the cell line and used in various concentrations from 0.25 mg/ml to 10 mg/ml. For immunofluorescence and western blotting, the cell lines were treated from 15 min to 96 h and 48 h, respectively, in a 37°C, 5% CO2 incubator.
Immunofluorescence
Each cell line was cultured in an 8-well chamber slide (Nunc™ Lab-Tek™) at a density of 0.3x105 cells/well, or a cover glass was placed in a 24-well cell culture plate and seeded at 0.5 × 105 cells/well. After 24 h, control human IgG, cHBIG, or lenvervimab were treated in a 5% CO2 incubator at 37°C for 15 min to 48 h, and then the cells were fixed with 3.7% paraformaldehyde for 15 min, followed by incubation for 15 min using 0.2% Triton X-100. Blocking was performed with 1% bovine serum albumin (BSA) for an hour. The primary antibodies used were mouse anti-HBsAg and mouse anti-Rab5 (early endosome marker), rabbit anti-LC3 (autophagy marker), mouse anti-calnexin (ER marker), mouse anti-giantin (Golgi marker), and Rab7 (multivesicular body marker) in a ratio of 1:100. For the corresponding secondary antibody, an antibody conjugated with fluorescence, goat anti-mouse IgG (488), goat anti-human IgG (568), and horse anti-rabbit IgG (568) were used in a ratio of 1:200. In addition, wheat germ agglutinin (633) and Alexa Fluor-phalloidin 488 were used in a ratio of 1:500. This immunofluorescence (IF) sample was observed using a Leica TCS SP8 confocal microscope, and the corresponding fluorescence was used.
Western Blot
Each cell line was seeded on a 6 well cell culture plate, treated with necessary IgG or reagents, and then the cells of each well were scraped and lysed on ice for 30 min after treatment with radioimmunoprecipitation assay lysis buffer and proteinase inhibitor. The lysed cells were centrifuged at 4°C for 30 min to obtain the supernatant. A 5x sample buffer was added, boiled for 5 min, and cooled on ice.
Western blotting was performed for 55 min at 130 V and 300 mA using 4–20% precast gel and then transferred to the polyvinylidene fluoride membrane at 80 V and 300 mA for 2 h at 4°C in a cold room. This was blocked with 5% BSA and washed with tris buffered saline. The primary antibody was incubated in a cold room at 4°C overnight, and the secondary antibody was incubated at room temperature for 1 h. The results were measured and analyzed using an LAS 4000 instrument.
As described in figure, each protein was loaded on a gel and transferred to one sheet of membrane. The proteins were detected using each antibody after cutting the membrane suitable size of the interested protein. In case of HBsAg, we used entire membrane to detect the various size of proteins consisting of HBsAg such as small, medium and large (Figure 4) (Supplementary file)
Cell culture medium protein assay
1) Western blot after polyethylene glycol (PEG) down
In order to characterize the proteins in the cell culture medium by Western blotting, the culture medium was collected separately and then centrifuged at 8000 rpm. The supernatant (2 ml of supernatant was mixed with 1 ml of 40% PEG, 400 µl of 4M NaCl, and DMEM to adjust to a total volume of 4 ml. This was mixed with a rotator at 4°C overnight, and the pellet was centrifuged at 4850 rpm at 4°C and used for Western blotting.
2) Enzyme-linked immunosorbent assay
The PLC/PRF/5 cell line was seeded into a 6 well plate. After 24 h,FBS free DMEM was added in each plate and, cHBIG or lenvervimab, and Fab portion of lenvervimab were added. and After 48 h, the supernatant was replaced with FBS-free DMEM. The supernatant was obtained every 12, 24, and 48 h, centrifuged at 10,000 rpm for 20 min at 4°C, and the supernatant was used for enzyme-linked immunosorbent assay (ELISA).
A human HBsAg ELISA kit was used. A blank sample was added to each well and incubated at 37°C for 90 min. After removing the liquids in the well and treating the supernatant with 100 µl of biotinylated detection antibody, it was gently tapped to mix well and incubated again at 37°C for 1 h. After washing each well three times with wash buffer, 100 µl of Horseradish peroxidase conjugate working solution was added and incubated at 37°C for 30 min. After washing each well five times with wash buffer, 90 µl of the substrate was treated and incubated at 37°C for 15 min. After 50 µl of the stop solution was added, the optical density was measured at 450 nm using a microplate reader.
Ethics
This study followed the ethical guidelines of the Declaration of Helsinki and was approved by the institutional review board of Seoul National University Hospital (institutional review board no 1401-081-550, 1701-004-819). In this study, we used human hepatocyte form patient sample after resection and we received informed consent with IRB approval (IRB number was described above). The dataset from qualified researchers trained in human subject confidentiality protocols may be sent to the Seoul National University Hospital institutional review board at [email protected] (https://cris.snuh.org).