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 - 0168-8278
VL - 45
SP - 547
EP - 552
JO - Journal of Hepatology
JF - Journal of Hepatology
IS - 4
ER -
