Study on in vitro model of hepatitis B virus-infected transwell chambers mediated by peripheral blood mononuclear cells

Abstract

Objective To observe the transport of hepatitis B virus (HBV)-infected peripheral blood mononuclear cells (PBMC) through placental barrier set up by choriocarcinoma trophoblast cells (Bewo cells), and to explore the biological role of PBMC as a carrier for HBV transport. Methods Bewo cells and PBMC were cultured and their proliferation and activity were detected by cell counting kit (CCK)-8. One hundred μL serum containing 5×106 copy/mL HBV DNA was used to infect PBMC, and cells infected with HBV were labeled by fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE). A co-culture model of Bewo cells and HBV-infected PBMC was set up by transwell chamber. The migration of HBV-infected PBMC was detected by flow cytometry. Realtime fluorescence quantitative polymerase chain reaction method was used to detect HBV DNA contents of PBMC under transwell chamber. Results PBMC and Bewo cells proliferated at around 24 h and entered into growth stagnation at around 120 h. The contents of PBMC labeled by green fluorescent at 0, 12, 24 and 48 h during co-culture under chamber were (0.445±0.021)%, (21.180±4.653)%, (34.830±7.156)% and (64.185±3.161)%, respectively. The amount of PBMC marked green fluorescence increased over prolonged incubation time (F=68.983, P=0.001). PBMC HBV DNA contents at 24 and 48 h of co-culture under chamber were (1.925±0.431)×103 copy/mL and (2.565±0.361)×103 copy/mL, respectively, indicating that PBMC under chamber were infected with HBV. Conclusions PBMC may be a target for HBV infection in extrahepatic tissues. Placental trophoblastic barrier built by transwell chambers may provide new ideas to investigate HBV transmission across the placenta in vitro. Key words: Hepatitis B virus; Peripheral blood mononuclear cells; Bewo cells; Transwell chamber