Cultivation of HepG2.2.15 on Cytodex-3: higher yield of hepatitis B virus and less subviral particles compared to conventional culture methods

TY - JOUR

T1 - Cultivation of HepG2.2.15 on Cytodex-3

T2 - higher yield of hepatitis B virus and less subviral particles compared to conventional culture methods

AU - Lupberger, Joachim

AU - Mund, Andreas

AU - Kock, Josef

AU - Hildt, Eberhard

PY - 2006/10

Y1 - 2006/10

N2 - BACKGROUND/AIMS: Several novel systems are available to study human hepatitis B virus (HBV) replication in cell culture demanding for efficient cell culture based systems for HBV production. The aim was to enhance HBV production of the HBV stably producing cell line HepG2.2.15 by cultivation on spherical micro substrate.METHODS: HepG2.2.15 was cultivated on microcarrier substrate Cytodex-3. HBV specific transcripts, viral protein and genome secretion, cell proliferation and MAP kinase signaling were analyzed. Infectivity of HBV particles was analyzed using primary tupaia hepatocytes.RESULTS: Compared to stationary flask cultures, HepG2.2.15 on Cytodex-3 secreted 18-fold more HBV genomes, more HBeAg per culture volume and less HBV surface antigen per extracellular viral genome equivalent. This was reflected by a significantly higher infectivity of supernatant derived from carrier grown HepG.2.2.15 cells tested by infection of primary tupaia hepatocytes. The amount of phosphorylated ERK-2 was significantly elevated in cells cultivated on microcarrier.CONCLUSIONS: The cultivation of HepG2.2.15 on Cytodex-3 increased production of infectious HBV particles and decreased secretion of subviral particles compared to the stationary cell cultivation. Microcarrier cultivation activates MAP kinase signaling that is crucial for HBV replication.

AB - BACKGROUND/AIMS: Several novel systems are available to study human hepatitis B virus (HBV) replication in cell culture demanding for efficient cell culture based systems for HBV production. The aim was to enhance HBV production of the HBV stably producing cell line HepG2.2.15 by cultivation on spherical micro substrate.METHODS: HepG2.2.15 was cultivated on microcarrier substrate Cytodex-3. HBV specific transcripts, viral protein and genome secretion, cell proliferation and MAP kinase signaling were analyzed. Infectivity of HBV particles was analyzed using primary tupaia hepatocytes.RESULTS: Compared to stationary flask cultures, HepG2.2.15 on Cytodex-3 secreted 18-fold more HBV genomes, more HBeAg per culture volume and less HBV surface antigen per extracellular viral genome equivalent. This was reflected by a significantly higher infectivity of supernatant derived from carrier grown HepG.2.2.15 cells tested by infection of primary tupaia hepatocytes. The amount of phosphorylated ERK-2 was significantly elevated in cells cultivated on microcarrier.CONCLUSIONS: The cultivation of HepG2.2.15 on Cytodex-3 increased production of infectious HBV particles and decreased secretion of subviral particles compared to the stationary cell cultivation. Microcarrier cultivation activates MAP kinase signaling that is crucial for HBV replication.

KW - Carcinoma, Hepatocellular

KW - Cell Count

KW - Cell Culture Techniques/methods

KW - Cell Line, Tumor

KW - Culture Media

KW - Dextrans

KW - Hepatitis B/virology

KW - Hepatitis B Surface Antigens/metabolism

KW - Hepatitis B e Antigens/metabolism

KW - Hepatitis B virus/growth & development

KW - Humans

KW - Liver Neoplasms

KW - MAP Kinase Signaling System

KW - Microspheres

KW - Mitogen-Activated Protein Kinase 1/metabolism

KW - Virion/growth & development

KW - Virulence

U2 - 10.1016/j.jhep.2006.05.012

DO - 10.1016/j.jhep.2006.05.012

M3 - Journal article

C2 - 16879893

SN - 0169-5185

VL - 45

SP - 547

EP - 552

JO - Journal of Hepatology, Supplement

JF - Journal of Hepatology, Supplement

IS - 4

ER -