title: An efficient system for secretory production of fibrinogen using a hepatocellular carcinoma cell line.
abstract: OBJECTIVE
  Despite an increasing demand, blood products are not always safe because most are derived from blood donations. One possible solution is the development and commercialization of recombinant fibrinogen, but this process remains poorly developed. This study aimed to develop an effective production system for producing risk-free fibrinogen using human hepatocellular cell lines and serum-free media.
  
  
  METHODS
  Three human liver cancer cell lines (HepG2, FLC-4 and FLC-7) were cultivated in a serum-supplemented medium or two serum-free media (ASF104N and IS-RPMI) to compare their fibrinogen secretion abilities. Fibrinogen subunit gene expression was estimated by quantitative polymerase chain reaction. Massive fibrinogen production was induced using a 5-mL radial flow bioreactor (RFB) while monitoring glucose metabolism. Subsequently, fibrinogen's biochemical characteristics derived from these cells were analyzed.
  
  
  RESULTS
  FLC-7 cell culture combined with IS-RPMI resulted in significantly better fibrinogen production (21.6 μg/10(7) cells per day). ASF104N had more positive effects on cell growth compared with IS-RPMI, whereas fibrinogen production was more efficient with IS-RPMI than with ASF104N. Changing the medium from ASF104N to IS-RPMI led to significantly increased fibrinogen gene expression and glucose consumption. In the RFB culture, the fibrinogen secretion rate of FLC-7 cells reached 0.73 μg/mL per day during a 42-day cultivation period. The subunit composition and clot formation activity of FLC-7 cell-derived fibrinogen corresponded to those of plasma fibrinogen.
  
  
  CONCLUSIONS
  The FLC-7 cell culture system is suitable for large-scale fibrinogen preparation production.
authors.affiliation: Department of Surgery, The Jikei University School of Medicine, Tokyo, Japan.|Department of Biochemistry, The Jikei University School of Medicine, Tokyo, Japan.
authors.forename: Michinori
authors.initials: M
authors.lastname: Matsumoto
authors.affiliation: Department of Laboratory Medicine, The Jikei University School of Medicine, Tokyo, Japan.
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Matsuura
authors.affiliation: Department of Biochemistry, The Jikei University School of Medicine, Tokyo, Japan.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Aoki
authors.affiliation: Department of Laboratory Medicine, The Jikei University School of Medicine, Tokyo, Japan.
authors.forename: Haruka
authors.initials: H
authors.lastname: Maehashi
authors.affiliation: Department of Core Research Facilities, The Jikei University School of Medicine, Tokyo, Japan.
authors.forename: Takeo
authors.initials: T
authors.lastname: Iwamoto
authors.affiliation: Department of Biochemistry, The Jikei University School of Medicine, Tokyo, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ohkawa
authors.affiliation: Department of Biochemistry, The Jikei University School of Medicine, Tokyo, Japan.
authors.forename: Kiyotsugu
authors.initials: K
authors.lastname: Yoshida
authors.affiliation: Department of Surgery, The Jikei University School of Medicine, Tokyo, Japan.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Yanaga
authors.affiliation: Department of Biochemistry, The Jikei University School of Medicine, Tokyo, Japan.|Division of Biology, Department of Natural Science, The Jikei University School of Medicine, Tokyo, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Takada
keywords: bioartificial liver; blood transfusion; blood-borne pathogens; fibrinogen; human cell line; radial flow bioreactor