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.
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.
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.
The FLC-7 cell culture system is suitable for large-scale fibrinogen preparation production.
© 2014 The Japan Society of Hepatology.

The mouse and human TPSB2 and TPSAB1 genes encode tetramer-forming tryptases stored in the secretory granules of mast cells (MCs) ionically bound to heparin-containing serglycin proteoglycans. In mice these genes encode mouse MC protease-6 (mMCP-6) and mMCP-7. The corresponding human genes encode a family of serine proteases that collectively are called hTryptase-β. We previously showed that the α chain of fibrinogen is a preferred substrate of mMCP-7. We now show that this plasma protein also is highly susceptible to degradation by hTryptase-β· and mMCP-6·heparin complexes and that Lys(575) is a preferred cleavage site in the protein α chain. Because cutaneous mouse MCs store substantial amounts of mMCP-6·heparin complexes in their secretory granules, the passive cutaneous anaphylaxis reaction was induced in the skin of mMCP-6(+)/mMCP-7(-) and mMCP-6(-)/mMCP-7(-) C57BL/6 mice. In support of the in vitro data, fibrin deposits were markedly increased in the skin of the double-deficient mice 6 h after IgE-sensitized animals were given the relevant antigen. Fibrinogen is a major constituent of the edema fluid that accumulates in tissues when MCs degranulate. Our discovery that mouse and human tetramer-forming tryptases destroy fibrinogen before this circulating protein can be converted to fibrin changes the paradigm of how MCs hinder fibrin deposition and blood coagulation internally. Because of the adverse consequences of fibrin deposits in tissues, our data explain why mice and humans lack a circulating protease inhibitor that rapidly inactivates MC tryptases and why mammals have two genes that encode tetramer-forming serine proteases that preferentially degrade fibrinogen.