Generation of large numbers of human dendritic cells from whole blood passaged through leukocyte removal filters: an alternative to standard buffy coats.
Many blood banks now use whole blood inline filtration to produce leukocyte-depleted blood products. As a result, a common source of large numbers of human dendritic cells (DC) for research purposes, namely standard buffy coats, has been lost. Therefore, we have adapted our conventional method for growing DC from CD14(+) precursors in order to make use of these filter units. A dextran solution containing human serum albumin was used to flush back the filters. After pelleting, mononuclear cells were obtained by standard density gradient centrifugation (Lymphoprep). To eliminate T cells, we used rosetting with sheep red blood cells. In addition to the classical PBMC, the cell population obtained after Lymphoprep centrifugation was found to contain high numbers of CD14(+) granulocytes which could be depleted by separation on an additional Percoll gradient. At this stage, FACS analysis revealed a cell population that resembled the CD14(+) monocyte-enriched population, obtained from traditional buffy coat preparations after Lymphoprep centrifugation and T cell elimination. Culture of the cells and the induction of maturation was identical to the previously described procedures, except that the culture time was reduced from 7 to 5 days and the maturation time from 3 to 2 days. Analyses of the major molecules indicative of DC maturation (CD83, CD86, CD208/DC-LAMP) and functional analyses of the T cell-stimulatory capacity of the DC population (using the MLR assay with normal peripheral T cells and naive T cells) revealed no major differences from buffy coat-derived DC preparations.
Ebner
S
S
Department of Dermatology, University of Innsbruck, Anichstrasse 35, A-6020, Innsbruck, Austria.
Neyer
S
S
Hofer
S
S
Nussbaumer
W
W
Romani
N
N
Heufler
C
C
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000145:classification / Q000166:cytology* / Q000276:immunology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology; D018950:Lipopolysaccharide Receptors; D007959:Lymphocyte Culture Test, Mixed; D013997:Time Factors
D018950:Lipopolysaccharide Receptors