Neutrophil precursor generation: effects of culture conditions

Abstract

The influence of feeding schedules on the expansion and differentiation of enriched PB CD34+ cells (84.9+/-14.7% purity) was studied after 12-13 days of serum-free liquid culture. CD34+ cell cultures were initiated (n=6) on day 0 (2 x 10(5) cells) in X-VIVO 10 medium containing 1% human albumin (HA) and 100 ng/ml each of rIL-3, rIL-6, rSCF, and rG-CSF. The cultures were supplemented on days 3, 6, and 9 as follows: condition 1, unfed (static culture); condition 2, 100 ng/ml rG-CSF; condition 3, split 1:2 medium + 100 ng/ml each rIL-3, rIL-6, rSCF, and rG-CSF; condition 4, split 1:2 medium + 100 ng/ml rG-CSF. The proliferative capacities (fold increase) of condition 2 (49.1+/-21.3), condition 3 (75.6+/-33.4), and condition 4 (63.1+/-23.8) cultures were significantly higher (p < 0.05) than that of the condition 1 unfed (35.5+/-14.0) cultures. Flow cytometric analysis (CD15-FITC/CD11b-PE) showed that the highest CD15+ cell purity (neutrophil precursors) was found in the condition 3 (1.18 x 10(7)+/-4.29 x 10(6)) cultures, followed by condition 4 (9.84 x 10(6)+/-3.57 x 10(6)), condition 2 (7.54 x 10(6)+/-2.06 x 10(6)), and condition 1 (4.78 x 10(6)+/-9.80 x 10(5)), respectively. The average cloning efficiency of the day 0 enriched CD34+ cells, 15.1%+/-10.3%, decreased to less than 0.2% in all of the day 12-13 cultures. These data suggest that feeding CD34+ cell cultures with rG-CSF alone, medium + rG-CSF, or medium + rIL3, rIL-6, rSCF, and rG-CSF enhances CD15+ neutrophil precursor (promyelocytes, myelocytes, metamyelocytes) production in vitro.