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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Wagner
Stephen J
SJ
American Red Cross Biomedical Services, Holland Laboratory, Blood and Cell Therapy Development, Rockville, Maryland 20855, USA. wagners@usa.redcross.org
Myrup
Andrew
A
Xu
Yanwen
Y
Walker
M Susan
MS
Welbon
Craig
C
Celluzzi
Christina M
CM
D001781:Blood Component Removal / Q000379:methods*; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D018925:Chemokines / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005312:Fetal Blood / Q000166:cytology*; D006801:Humans; D008214:Lymphocytes; D009000:Monocytes / Q000166:cytology* / Q000378:metabolism; D010587:Phagocytosis / Q000276:immunology
D018925:Chemokines; D016207:Cytokines; D015232:Dinoprostone