Effects of synchronization on CD40 expression and antibody production in hybridoma cells stimulated with anti-mIgG.
In previous experiments with the 55-6 hybridoma cell line, we showed that cell stimulation with anti-mouse surface immunoglobulin G antibody (anti-mIgG) increased both CD40 expression and specific monoclonal antibody (mAb) production rate. Cell preincubation with lipopolysaccharide (LPS) prior to anti-mIgG stimulation enhanced these results. Moreover, the expression of both CD40 and surface immunoglobulin G (sIgG) were higher for cells in the G1 phase of the cell cycle. Therefore, to determine the relationship between cell cycle position, CD40 expression, and mAb productivity, in this work cells were synchronized in the G1 phase by thymidine block. In addition, synchronized cells were subjected to different treatments with anti-mIgG. Although synchronized cells showed a slight increase in both CD40 expression and maximum specific growth rate (mu max) compared with unsynchronized cells, specific productivity did not show significant changes. However, the stimulation of synchronized cells with anti-mIgG increased over 65% the expression of CD40 and over 50% the specific productivity in comparison with that obtained on unsynchronized cells after anti-mIgG stimulation. These data improved additionally over 15 and 60%, respectively, by adding 2 mM thymidine to the culture medium. These results suggest that the effect of the positive association between G1 phase, CD40 expression, and specific productivity is subordinated to the effect of anti-mIgG stimulation, which is enhanced by increasing the percentage of cells on the G1 phase of the cell cycle. Contrary to expectations, LPS preincubation of synchronized cells prior to anti-mIgG stimulation did not increase the specific productivity in comparison with non-preincubated cells, and the expression of CD40 was minor compared to that on non-preincubated cells.
Martín-López
Alicia
A
Department of Chemical Engineering, University of Almería, 04120 Almería, Spain.
García-Camacho
Francisco
F
Contreras-Gómez
Antonio
A
Molina-Grima
Emilio
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D015703:Antigens, CD / Q000096:biosynthesis; D018941:Antigens, CD19 / Q000096:biosynthesis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000096:biosynthesis; D001709:Biotechnology / Q000379:methods*; D019013:CD40 Antigens / Q000096:biosynthesis*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D016193:G1 Phase; D006825:Hybridomas / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry*; D037181:Lectins, C-Type; D008070:Lipopolysaccharides / Q000737:chemistry; D051379:Mice; D013936:Thymidine / Q000737:chemistry; D000099192:CD69 Antigens
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018941:Antigens, CD19; D000945:Antigens, Differentiation, T-Lymphocyte; D019013:CD40 Antigens; D007074:Immunoglobulin G; D037181:Lectins, C-Type; D008070:Lipopolysaccharides; D013936:Thymidine; D000099192:CD69 Antigens