NEUTROPHIL PRECURSOR GENERATION - EFFECTS OF CULTURE CONDITIONS

The influence of feeding schedules on the expansion and differentiaton 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 init iated (n = 6) on day 0 (2 x 10(5) cells) in X-VIVO 10(TM) medium conta ining 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 foll ows: condition 1, unfed (static culture); condition 2, 100 ng/ml rG-CS F; 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 prol iferative capacities (fold increase) of condition 2 (49.1 +/- 21.3), c ondition 3 (75.6 +/- 33.4), and condition 4 (63.1 +/- 23.8) cultures w ere significantly higher (p < 0.05) than that of the condition 1 unfed (35.5 +/- 14.0) cultures. Flow cytometric analysis (CD15-FITC/CD11b-P E) showed that the highest CD15+ cell purity (neutrophil precursors) w as 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 enr iched 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.