Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells

Although it has also been shown to infect extrahepatic organs including the kidney and testis, HBV typically infects human hepatocytes. Nevertheless, hepatoma cell lines (such HepG2 and Huh7) that overexpress sodium taurocholate co-transporting polypeptide (NTCP), a functional HBV receptor, are the only ones that can be used in cell-based HBV models. This procedure presents HepG2-NE (HepG2 overexpressing NTCP) and 293T-NE-3NRs (HNF4α, RXRα, PPARα, and human NTCP overexpressed) as model cell lines. Either concentrated HepG2.2.15 HBV virus particles or co-culturing HepG2.2.15 with the target cell lines are used to infect these cell lines with HBV. HBV infection is confirmed by HBcAg immunofluorescence staining. Our investigation of HBV infection in non-hepatic cell lines will be aided by these two techniques.

Experimental Materials and Recommended Services

Name	Comments	Related Products and Services
293T-NE	Cell model for HBV infection	Cas9 Stable Cell Line - HepG2 GFP Stable Cell Line-HepG2 RFP Stable Cell Line-HepG2 Luciferase Reporter Cell Line - HepG2 GFP/Luc Reporter Cell Line - HepG2 Custom Cell Lines Services
293T-NE-3NRs	Cell model for HBV infection	SLC10A1 miRNA 3'UTR clone Panoply™ Human SLC10A1 Over-expressing Stable Cell Line
594 labeled goat against rabbit IgG	Filter for HepG 2.2.15 supernatant	Antibody-Producing Cell Lines Development Antibody Conjugate Kit Knockout Cell Lines in Antibody Screening and Validation
HBcAg antibody	For immunofluorescence assay, primary antibody
HepG2-NE	For immunofluorescence assay, a second antibody

Procedure

In accordance with national biosafety rules, culture HepG2.2.15 cells and manage HBV infection in biosafety level II (P2) or biosafety level III (P3) laboratories. Make sure that all workers are vaccinated against HBsAb and tested for the virus before beginning any HBV studies. Continually check the state of the cells. Utilize human serum samples that have been authorized by the Institutional Review Board.

Fig 1 illustrates a comprehensive protocol for infecting novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4α, RXRα, and PPARα) and traditional hepatic cells (HepG2-NE, expressing human NTCP) with Hepatitis B virus (HBV). (doi: 10.3791/61414) Figure 1. Illustrates the protocol. HepG2.2.15 cells were co-cultured with 293T-NE-3NRs, and infection was observed using immunofluorescence microscopy. Alternatively, the supernatant containing viral particles was concentrated and introduced into cultured 293T-NE-3NRs cells. (Sun, X., et al., 2020)

1. HepG2.2.15 cell culture:

a. Dispose of all materials contacting HepG2.2.15 cells, including supernatant and associated items, after overnight soaking in 2% virucide.

b. Thaw HepG2.2.15 cells by gently swirling in a 37°C water bath, keeping the cap dry to prevent contamination. Thaw rapidly (~2 min).

c. Disinfect the vial with 70% ethanol after thawing and transfer cells to a 25 cm2 tissue culture flask. Add 4 mL complete culture medium (DMEM + 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin). Incubate at 37°C, 5% CO₂.

2. Collection of HepG2.2.15 cell culture supernatant:

a. Culture HepG2.2.15 cells in T25 flasks at 37°C, 5% CO₂. Change the medium every 3 days.

b. Collect supernatant in a 50 mL centrifuge tube, seal tightly, and wrap with paraffin film.

c. Store at -20°C to maintain HBV activity.

3. Concentrating HepG2.2.15 supernatant:

a. Thaw supernatant at 4°C, then filter with a 0.45 µm membrane to remove cell fragments.

b. Add filtered supernatant to a virus concentrator column, centrifuge, and collect concentrate.

c. Wash the column with DMEM, then collect. Store concentrate at -80°C.

4. Detection of HBV DNA in supernatant concentrate:

a. Dilute concentrate 20-fold, prepare PCR master mix and Taq polymerase.

b. Perform real-time PCR using appropriate conditions and generate a standard curve.

c. Calculate HBV DNA concentration and store concentrate at -80°C.

5. In vitro infection of HepG2-NE and 293T-NE-3NRs cells:

a. Prepare infection medium, add gelatin to plate wells, and seed cells.

b. Incubate cells for 24 h, observe and proceed with infection if healthy.

c. Prepare infection complex, add to wells, and incubate for 24 h.

d. Wash cells and change the medium every 2 days.

6. Co-culture HepG2.2.15 with HepG2-NE/293T-NE-3NRs/293T-NE:

a. Prepare plate wells, and seed cells, and incubate for 24 h.

b. After 24 h, co-culture by placing HepG2.2.15 inserts into wells with other cells. Incubate and change medium every 3-4 days.

c. After 10 days, remove inserts, wash wells, and fix cells.

7. Perform immunofluorescence for HBcAg:

a. Fix cells with ice-cold methanol, and wash with PBS.

b. Add BSA, apply HBcAg primary antibody, and incubate overnight.

c. Wash wells with PBST, add a second antibody solution, and incubate.

d. Wash wells with PBST, add DAPI, and observe under a microscope.

This method introduces protocols for HBV infection in non-hepatic 293T-NE-3NRs and hepatoma-based HepG2-NE cells. Despite obstacles like cell fragility, crucial procedures like prompt medium changes and delicate cell washing guarantee effective infection. This technology complements conventional techniques by providing a physiologically appropriate model for HBV infection through the use of modified 293T-NE-3NRs cells.

* For research use only. Not intended for any clinical use.

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