The establishment of a combined serum-free and serum-supplemented culture method of obtaining functional cord blood-derived human mast cells.
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 FcvarepsilonRI alpha and released >30% of the histamine content upon anti-IgE stimulation than those cultured without serum.


CONCLUSIONS
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.
Dahl
Christine
C
A Research, Department of Pediatrics, Aarhus University Hospital, Brendstrupgaardsvej 100, 8200 Aarhus C, Denmark. christine.dahl@iekf.au.dk
Saito
Hirohisa
H
Nielsen
Henrik Vendelbo
HV
Schiøtz
Peter Oluf
PO
D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D015550:Cell Degranulation / Q000276:immunology; D002455:Cell Division; D002469:Cell Separation; D002470:Cell Survival; D016895:Culture Media, Serum-Free; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D006801:Humans; D008407:Mast Cells / Q000166:cytology* / Q000276:immunology; D011247:Pregnancy; D017455:Receptors, IgE
D016895:Culture Media, Serum-Free; D017455:Receptors, IgE