BACKGROUND: DC generated from monocytes have been used for vaccines. We have developed a monocyte enrichment procedure by depleting T and B cells with anti-CD2 and anti-CD19 Abs using the automated Isolex 300i magnetic cell selector for clinical-scale DC generation in gas permeable SteriCell culture bags. We have also compared DC function, yield and purity of DC generated from adherent monocytes using culture bags in a closed system, with DC generated in conventional tissue culture flasks. METHODS: Monocytes were enriched from normal donor apheresis products using CD2/19 depletion with experimental software on the Isolex 300i (ISO), adherence (AD) to SteriCell bags and to T175 flasks and then cultured for 7 days in serum-free X-VIVO 15 media with GM-CSF and IL-4. Phenotype and dextran uptake were analyzed by flow cytometry and allogeneic MLR was also evaluated. RESULTS: ISO-DC and AD-DC from SteriCell bags showed similar viability. Higher purity of ISO-DC than AD-DC was measured by forward- and side-scatter flow cytometry. Similar expression of CD1a, CD80, CD86 and CD83 were observed in both ISO-DC and AD-DC. Similar dextran uptake and allo MLR were also observed. DISCUSSION: These data indicated that functional DC were generated in gas permeable SteriCell culture bags from both ISO- and AD-monocytes in a closed system.
BACKGROUND: DC generated from monocytes have been used for vaccines. We have developed a monocyte enrichment procedure by depleting T and B cells with anti-CD2 and anti-CD19 Abs using the automated Isolex 300i magnetic cell selector for clinical-scale DC generation in gas permeable SteriCell culture bags. We have also compared DC function, yield and purity of DC generated from adherent monocytes using culture bags in a closed system, with DC generated in conventional tissue culture flasks. METHODS: Monocytes were enriched from normal donor apheresis products using CD2/19 depletion with experimental software on the Isolex 300i (ISO), adherence (AD) to SteriCell bags and to T175 flasks and then cultured for 7 days in serum-free X-VIVO 15 media with GM-CSF and IL-4. Phenotype and dextran uptake were analyzed by flow cytometry and allogeneic MLR was also evaluated. RESULTS: ISO-DC and AD-DC from SteriCell bags showed similar viability. Higher purity of ISO-DC than AD-DC was measured by forward- and side-scatter flow cytometry. Similar expression of CD1a, CD80, CD86 and CD83 were observed in both ISO-DC and AD-DC. Similar dextran uptake and allo MLR were also observed. DISCUSSION: These data indicated that functional DC were generated in gas permeable SteriCell culture bags from both ISO- and AD-monocytes in a closed system.
Authors: Timo Buhl; Tobias J Legler; Albert Rosenberger; Anke Schardt; Michael P Schön; Holger A Haenssle Journal: Cancer Immunol Immunother Date: 2012-04-22 Impact factor: 6.968
Authors: Alexander Michael Dohnal; Sebastian Graffi; Volker Witt; Christina Eichstill; Dagmar Wagner; Sidrah Ul-Haq; Doris Wimmer; Thomas Felzmann Journal: J Cell Mol Med Date: 2008-03-17 Impact factor: 5.310