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Updated: Aug 17, 2026
Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood
Published on: December 23, 2015
Toward closed-system culture of blood origin endothelial cells
Stephen J Wagner1, Andrew C Myrup
1Blood Components Department, American Red Cross Holland Laboratory, Rockville, Maryland 20855, USA. wagners@usa.redcross.org
Background:
Blood outgrowth endothelial cells (BOECs) are thought to arise from very rare progenitors that are present in the mononuclear fraction of marrow or peripheral blood. Recently, BOECs have been expanded from progenitors present in buffy coat into confluent monolayers on fibronectin- or collagen-coated polystyrene surfaces. A method for sterile closed-system culture of these cells has not been described, however. Here, efforts are described toward developing closed-system culture of BOECs derived from progenitors present in a mononuclear apheresis unit by use of a cord blood filter, a sterile connection device, and a fibronectin-coated polycarbonate cassette.
Study Design And Methods:
Strongly adherent cells from a mononuclear apheresis unit were eluted from a cord blood filter and resuspended in EGM-2 with 10 percent serum. Approximately 2 x 10(8) eluted cells were introduced into human fibronectin-coated polycarbonate cassettes. Medium was introduced and removed from cassettes with a sterile connection device and changed every 2 days. After expansion, cells were either cryopreserved or characterized by fluorescence-activated cell sorting analysis and ability to take up Dil-Ac-LDL.
Results:
After 2 to 3 weeks of culture, 3 to 28 colonies with cobblestone morphology were observed in cassettes and passed to new cassettes within 3 to 4 weeks. By approximately 5 weeks of culture, 2 x 10(6) cells were typically obtained. BOECs uniformly took up Dil-Ac-LDL and were CD31+, CD105+, CD146+, CD45-, and CD14-. A population of BOECs was HLA-ABC+ or CD34+.
Conclusion:
BOEC progenitors can be isolated from mononuclear apheresis units with cord blood filters, expanded with fibronectin-coated polycarbonate cassettes, and cryopreserved.
