Monocyte enrichment of mononuclear apheresis preparations with a multistep back‐flush procedure on a cord blood filter

Abstract

Monocytes or mononuclear cells have been investigated for the treatment of chronic wounds and spinal cord injuries, as well as serve as a source for dendritic or endothelial cell culture. Because these cells may have clinical benefit yet no rapid and inexpensive closed system for monocyte purification is commercially available, a method was investigated to enrich monocytes from mononuclear apheresis units using a cord blood filter. A 4-step method for monocyte enrichment was developed which involved 1) filtering a mononuclear apheresis unit through a cord blood filter, 2) chasing with medium to remove non-adherent residual cells and plasma, 3) back-flushing under low shear conditions to remove loosely adherent lymphocytes, and 4) back-flushing under high shear conditions to collect a fraction enriched in monocytes. Apheresis units and enriched monocyte preparations were characterized by cell count and differential, filter-isolated preparations were cryopreserved, and thawed preparations were assayed for viability, and phagocytosis. Enriched monocyte preparations were also assayed for inflammatory cytokines secretion and secretion of prostaglandin E2 during short-term culture. Monocytes were viable, capable of phagocytosis, and enriched using the multistep filter elution technique to represent 42 +/- 13-percent of white cells in the final preparation. Fifty-three-percent of monocytes were recovered in the final preparation, while total cell counts of red cells, platelets, neutrophils and lymphocytes were reduced to 3.0, 3.0, 4.5 and 16-percent, respectively, from levels present in mononuclear apheresis units. Filter enriched monocyte preparations secreted IL-8, IL-6, MCP-1, and MIP-1alpha, during short term culture. The use of a multi-step back flush procedure with a cord blood filter resulted in rapid enrichment of viable and functional monocytes from mononuclear apheresis units with significant reduction of contaminating platelets and red cells.