Development of a closed-system process for clinical-scale generation of DCs: evaluation of two monocyte-enrichment methods and two culture containers.
BACKGROUND
Clinical immunotherapy trials using DCs depend on large-scale methods for DC generation that fulfil current good manufacturing practice requirements. Our goal was to develop data on two variables, monocyte-enrichment method and culture container, which could be used to design a closed-system process for ex vivo generation of immature DCs.


METHODS
Mononuclear cells were collected by leukapheresis and enriched for monocytes by either counterflow centrifugal elutriation, or immunomagnetic selection using Isolex, an automated closed-system device. Monocytes were cultured for 7 days in serum-free medium with GM-CSF and IL-4, using either plastic flasks or gas-permeable Stericell bags. Monocytes and cultured DCs were evaluated for yield, flow cytometric phenotype, and in vitro function in MLR, and autologous recall responses to tetanus toxoid and influenza virus.


RESULTS
Enriched monocyte products from elutriation and immunomagnetic selection were equivalent in yield and purity, and were capable of generating immature DCs in either flasks or bags. DCs from all four culture conditions were equivalent in yield, phenotype, and in vitro function. Mean DC yield was 67-80% per seeding monocyte, and 11-13% per starting mononuclear cell (MNC). A leukapheresis product containing 5 x 10(9) MNCs processed by this method could therefore yield approximately 5 x 10(8) immature DCs.


CONCLUSIONS
In this manufacturing process, the Isolex system was equivalent to elutriation, and Stericell bags were equivalent to flasks. Together, the Isolex system and Stericell bags can be incorporated into a closed-system process to generate immature DCs.
Wong
E C C
EC
Department of Transfusion Medicine, Warren G Magnusen Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA.
Lee
S M
SM
Hines
K
K
Lee
J
J
Carter
C S
CS
Kopp
W
W
Bender
J
J
Read
E J
EJ
D019264:Adoptive Transfer / Q000379:methods*; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology / Q000637:transplantation; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods; D007937:Leukapheresis / Q000379:methods; D007959:Lymphocyte Culture Test, Mixed / Q000379:methods; D008565:Membrane Proteins / Q000032:analysis; D009000:Monocytes / Q000166:cytology* / Q000502:physiology; D013234:Stem Cells / Q000502:physiology
D016895:Culture Media, Serum-Free; D008565:Membrane Proteins