Survival of hBcl-2 Transgenic B Cells in Culture

Wild-type B cells rapidly die in culture. Hence, we sought to define specific conditions that would allow the sustained survival of B cells for signaling studies. We compared several alternative potential primary cell signaling models, specifically hbcl-2 transgenic B cells and wild-type B cells stimulated with BAFF or anti-CD40 survival promoting ligands. Initial experiments using defined serum-free medium showed that after three days in culture, the survival of hbcl-2 transgenic B cells was 80% of the initial cells plated (Fig. 1). In contrast, the survival of wild-type B cells was less than 10%. Stimulation of wild-type B cells with BAFF or anti-CD40 increased viability to 37% and 43%, respectively (Fig. 1). From these results, we determined that the survival of hbcl-2 transgenic B cells was superior to that of either BAFF- or anti-CD40-stimulated wild-type B cells. Moreover, BAFF or anti-CD40-stimulated wild-type B cells were partly activated and more proliferative (data not shown), and yet the percentage of viable cell numbers remained relatively low (only 37% to 43%), such that assays performed with these populations contained significant numbers of dead or dying cells as well as cell debris. Therefore, wild-type B cells stimulated with BAFF and anti-CD40 were not investigated further as potential cultured cell models for studying primary cell signaling.

Fig. 1. Cell viability during culture of hbcl-2 transgenic and wild-type B cells. Splenic B cells were cultured in 6- or 24-well Costar ultralow plates at a density of 6 x 106/ml, with 3 ml or 0.6 ml/well, respectively, in serum-free medium (SIMDM). Wild-type B cells were stimulated with BAFF (3.7 nM = 62.5 ng/ml) or hamster IgM anti-CD40 (4.2 nM = 0.63 mg/ml) as indicated (anti-CD40 isotype control antibody had no significant effect on cell survival, data not shown). After one or three days in culture, cells were harvested, and viable cells were counted using trypan blue exclusion. The average viability data (+SD) for four experiments for each strain or treatment group is shown.

We then extended the period of culture of hbcl-2 transgenic B cells, comparing survival in serum-free medium to survival in medium supplemented with 10% fetal bovine serum (FBS). FBS improved survival: the survival of hbcl-2 transgenic B cells after six days of culture without serum was approximately 40%, but the survival with 10% FBS was approximately 80% (Fig. 2).

Fig. 2. Human bcl-2 transgenic B cell viability in culture -/+10% FBS. Human bcl-2 transgenic B cells were cultured at 6 x 106/ml in 24-well plates, using 0.6 ml/well SIMDM with or without 10% FBS added. On days 3 and 6, half of the culture medium was removed and replaced with fresh medium. On days 1, 3, 6, and 9, wells were harvested, and viable cells were counted after testing for trypan blue exclusion. The graphs show standard deviations for points where n = 3 experiments (i.e., three separate cultures of hbcl-2 transgenic B-cell preparations); for other points n = 2.