Abstract
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 initiated (n = 6) on day 0 (2 X 105 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 107 ± 4.29 X 106) cultures, followed by condition 4 (9.84 X 106 ± 3.57 X 106), condition 2 (7.54 X 106 ± 2.06 X 106), and condition 1 (4.78 X 106 ± 9.80 X 105), 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.
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