Toward closed-system culture of blood origin endothelial cells.
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.


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+.


CONCLUSIONS
BOEC progenitors can be isolated from mononuclear apheresis units with cord blood filters, expanded with fibronectin-coated polycarbonate cassettes, and cryopreserved.
Wagner
Stephen J
SJ
Blood Components Department, American Red Cross Holland Laboratory, Rockville, Maryland 20855, USA. wagners@usa.redcross.org
Myrup
Andrew C
AC
D015703:Antigens, CD; D018952:Antigens, CD34; D015415:Biomarkers / Q000097:blood; D001781:Blood Component Removal; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D015925:Cryopreservation; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism; D000071063:Endoglin; D004730:Endothelium, Vascular / Q000166:cytology* / Q000276:immunology; D005312:Fetal Blood / Q000166:cytology; D005353:Fibronectins / Q000378:metabolism; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology / Q000502:physiology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism; D018919:Neovascularization, Physiologic; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011956:Receptors, Cell Surface; D019947:Receptors, Interleukin-6; D013997:Time Factors; D019010:Vascular Cell Adhesion Molecule-1
D015703:Antigens, CD; D018952:Antigens, CD34; D015415:Biomarkers; D003470:Culture Media; C064206:ENG protein, human; D000071063:Endoglin; D005353:Fibronectins; D005456:Fluorescent Dyes; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011956:Receptors, Cell Surface; D019947:Receptors, Interleukin-6; D019010:Vascular Cell Adhesion Molecule-1; D016650:Fluorescein-5-isothiocyanate