Development of a clinical grade procedure for generation of mRNA transfected dendritic cells from purified frozen CD34(+) blood progenitor cells.
Enriched CD34(+) peripheral blood progenitor cells (PBPC) are frequently used as stem cell support in cancer patients following high dose therapy. Since precursor dendritic cells (DCs) originate from haematopoietic progenitor cells, purified CD34(+) cells might also serve as starting cells for ex- vivo production of DC. In the present study we developed a clinical grade procedure for ex- vivo production of DC derived from enriched CD34(+) cells. Different concentrations of CD34(+) cells were grown in gas-permeable Teflon bags with different serum-free and serum-containing media supplemented with GM-CSF, IL-4, TNF-alpha, SCF, Flt-3L and INF-alpha. Serum-free CellGroSCGM medium for 7 days followed by CellGroDC medium in 7 days gave the same results as serum-containing medium. After incubation the cultured cells containing immature DCs were concentrated and transfected with tumour mRNA from human prostate cancer cell lines employing a highly efficient electroporation procedure. Thawed transfected DCs were able to elicit primary T-cell responses in vitro against antigens encoded by the prostate cancer mRNA as shown by ELISPOT assay using mock-transfected DCs as control. Our results show that frozen enriched CD34(+) cells can be an alternative and efficient source for production of DCs for therapeutic purpose.
Laboratory for Cellular Therapy, Norwegian Radium Hospital, University of Oslo, Oslo, Norway.
L J
LJ
Mu
P
P
Lazarova
G
G
Gaudernack
S
S
Saeboe-Larssen
G
G
Kvalheim
D018952:Antigens, CD34 / Q000378:metabolism*; D045744:Cell Line, Tumor; D002469:Cell Separation; D015925:Cryopreservation; D003470:Culture Media; D016207:Cytokines / Q000378:metabolism; D003713:Dendritic Cells / Q000378:metabolism* / Q000637:transplantation*; D006133:Growth Substances / Q000378:metabolism; D006801:Humans; D008297:Male; D009000:Monocytes / Q000378:metabolism / Q000637:transplantation; D010641:Phenotype; D011471:Prostatic Neoplasms; D012333:RNA, Messenger / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000378:metabolism; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000378:metabolism*; D014162:Transfection
D018952:Antigens, CD34; D003470:Culture Media; D016207:Cytokines; D006133:Growth Substances; D012333:RNA, Messenger; D011994:Recombinant Proteins