Purification of hematopoietic cells often includes an adherence step for the removal of monocytes. Recent studies have shown that some hematopoietic cells are adherent and that plastic adhesion might activate monocyte/macrophage for cytokine production. We investigated the possibility of using L-phenylalanine methyl ester (PME) as an alternative approach to monocyte removal, particularly for large-scale cell separations. Cell yield, extent of monocyte removal and cloning efficiency of colony-forming progenitor cells were compared to that produced by plastic adhesion. Starting with mononuclear cells (MNC) from adult peripheral blood, cell recovery after PME treatment was higher than that achieved with adherence and fewer monocytes were present. Cloning efficiency of the granulocyte-monocyte colony-forming progenitor cells (CFU-gm) was not affected by the PME treatment. PME treatment and plastic adhesion of low-density cord blood cells produced comparable cell recovery, monocyte depletion and cloning efficiencies for CFU-gm and burst forming units for erythroid progenitor cells (BFU-e). The PME procedure was optimized for the purification of hematopoietic cells from large quantities (less than 1 x 10(9)) of low density. T cell-depleted peripheral blood MNC containing 60% to 85% monocytes. Compared to two cycles of plastic adhesion, PME treatment permitted higher cell recovery (5.9% vs. 1.3%), lower monocyte contamination (4.5% vs. 10.7%) and substantial cost, time and labor savings without compromising CFU-gm growth.

SUMMARY:Methods for culturing hematopoietic colonies from human peripheral blood are described in this report.Granulocyte-macrophage progenitor cells (CFU-GM: colony forming units-granulocyte/macrophage) are grown in 35-ram dishes containing 4 ml of 0.35% agarose in Iscove's modified Dulbecco's medium (IMDM) with 20% prescreened fetal bovine serum (FBS). Colony-stimulating activity is provided by leukocyte-conditioned medium, and nonactivated autologous T lymphocytes or recombinant granulocyte-and granulocyte/macrophage-colony stimulating factor or both. This procedure minimize the growth inhibition caused by monocytes. Erythroid progenitor cells (BFUe: burst forming units-erythroid) are cultured in 1 ml of 0.35% agarose in IMDM with 30% FBS and conditioned medium from human bladder carcinoma cell line 5637 or recombinant interleukin 3. Both assays have proved useful in studying the enrichment of peripheral blood hematopoietic cells.