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Due to the wide range of methods for generating DCs, there is no common protocol or defined set of criteria to validate the immunogenicity and function of DC vaccines.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Monocyte-derived DCs were generated during 4 days of culture with recombinant granulocyte-macrophage colony stimulating factor and interleukin-4, and pulsed with tumor lysate produced by hypochlorous acid oxidation of tumor cells. Different culture parameters for clinical-scale DC preparation were investigated, including: 1) culture media; 2) culture surface; 3) duration of activating DCs with lipopolysaccharide (LPS) and interferon (IFN)-gamma; 4) method of DC harvest; and 5) cryomedia and final DC product formulation.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>DCs cultured in CellGenix DC media containing 2% human AB serum expressed higher levels of maturation markers following lysate-loading and maturation compared to culturing with serum-free CellGenix DC media or AIM-V media, or 2% AB serum supplemented AIM-V media. Nunclon\u2122\u0394 surface, but not Corning<jats:sup>\u00ae<\/jats:sup>tissue-culture treated surface and Corning<jats:sup>\u00ae<\/jats:sup>ultra-low attachment surface, were suitable for generating an optimal DC phenotype. Recombinant trypsin resulted in reduced major histocompatibility complex (MHC) Class I and II expression on mature lysate-loaded DCs, however presentation of MHC Class I peptides by DCs was not impaired and cell viability was higher compared to cell scraping. Preservation of DCs with an infusible cryomedia containing Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin, and DMSO yielded higher cell viability compared to using human AB serum containing 10% DMSO. Finally, activating DCs for 16 hours with LPS and IFN-\u03b3 stimulated robust mixed leukocyte reactions (MLRs), and high IL-12p70 production<jats:italic>in vitro<\/jats:italic>that continued for 24 hours after the cryopreserved DCs were thawed and replated in fresh media.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>This study examined criteria including DC phenotype, viability, IL-12p70 production and the ability to stimulate MLR as metrics of whole oxidized tumor lysate-pulsed DC immunogenicity and functionality. Development and optimization of this unique method is now being tested in a clinical trial of autologous oxidized tumor lysate-pulsed DC in clinical-scale in recurrent ovarian, primary peritoneal or fallopian tube cancer (NCT01132014).<\/jats:p><\/jats:sec>","DOI":"10.1186\/1479-5876-9-198","type":"journal-article","created":{"date-parts":[[2011,11,16]],"date-time":"2011-11-16T03:27:48Z","timestamp":1321414068000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":25,"title":["Optimizing parameters for clinical-scale production of high IL-12 secreting dendritic cells pulsed with oxidized whole tumor cell lysate"],"prefix":"10.1186","volume":"9","author":[{"given":"Cheryl L-L","family":"Chiang","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dawn A","family":"Maier","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lana E","family":"Kandalaft","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andrea L","family":"Brennan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Evripidis","family":"Lanitis","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qunrui","family":"Ye","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bruce L","family":"Levine","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Brian J","family":"Czerniecki","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daniel J","family":"Powell Jr","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"George","family":"Coukos","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2011,11,14]]},"reference":[{"key":"975_CR1","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/S0022-1759(98)00208-7","volume":"223","author":"B Thurner","year":"1999","unstructured":"Thurner B, R\u00f6der C, Dieckmann D, Heuer M, Kruse M, Glaser A, Keikavoussi P, K\u00e4mpgen E, Bender A, Schuler G: Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application. 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