The establishment of a combined serum-free and serum-supplemented culture method of obtaining functional cord blood-derived human mast cells

Abstract

Background: Serum-free cultures supplemented with stem cell factor (SCF) and IL-6 is reported to support the extensive growth of less functional human cord blood-derived mast cells. Objective: To obtain more functional mast cells from cord blood, we developed a culture system combining a serum-free condition for 0–8 weeks of culture, and followed by a serum-supplemented culture condition and examined the function of the cells compared to the cells cultured continuously in serum-free condition. Methods: Human cord blood progenitors were purified with anti-CD133 antibody. They were cultured in a serum-free medium StemSpan supplemented with SCF at 100 ng/ml and IL-6 at 50 ng/ml for 8 weeks. Then, an aliquot of the cultured cells were cultured in the above condition but further supplemented with 10% fetal calf serum (FCS). Results: The addition of FCS after 8 weeks of culture significantly increased the amount of histamine per mast cell (3.8 pg/cell) when compared to the serum-free condition (0.7 pg/cell). The cells cultured with FCS after 8 weeks expressed more FcεRI α and released >30% of the histamine content upon anti-IgE stimulation than those cultured without serum. Conclusion: It is uncertain why FCS enhanced the functional maturation of mast cells when added after week 8 of culture but suppressed mast cell development when added at day 0 of culture. Yet, the present method combining a serum-free culture system with a serum-supplemented culture system seems to be beneficial for most of the laboratories to obtain functional human mast cells.