A closed and single-use system for monocyte enrichment: potential for dendritic cell generation for clinical applications.
ABSTRACT This study evaluated the ability of a modified cell separator (Cobe Spectra Apheresis) system to isolate monocytes (MOs) by elutriation. The evaluation was performed in two independent international laboratories. The capacity of collected MOs to differentiate into dendritic cells (DCs) was also assessed.
MNCs from platelet apheresis residues were elutriated on a modified cell separator (Cobe Spectra Apheresis system) using a custom disposable set. Cells were separated according to their size and density. Recovery and purity of the collected cell product were evaluated by impedance counting and flow cytometry. DCs were differentiated in culture from the elutriated MOs and characterized by their surface markers and stimulatory capacity in a mixed WBC reaction assay.
Six apheresis mononuclear cell products were used by each laboratory. The separation was achieved in less than 1 hour. Collected MOs had the potential to differentiate into DCs.
The modified cell separator is an easy and fast device to obtain highly enriched MOs with a DC differentiation potential. The system is closed and employs a single-use disposable set and is more amenable to good tissue practice. This method could become a valuable tool for DC-based active immunotherapy.
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ABSTRACT: Clinical-scale lymphocyte enrichment from a leukapheresis product has been performed most routinely using costly magnetic bead separation systems that deplete monocytes, but this procedure may leave behind residual beads or antibodies in the enriched cell product. Counterflow centrifugal elutriation has been demonstrated previously to enrich monocytes efficiently for generation of dendritic cells. This study describes a modified elutriation procedure for efficient bead-free economical enrichment of lymphocytes from leukapheresis products from healthy donors and study subjects with human immunodeficiency virus (HIV) infection or malignancy. Modified program settings and conditions for the CaridianBCT Elutra device were investigated to optimize lymphocyte enrichment and recovery. Lymphocyte enrichment was measured using a novel approach utilizing cell sizing analysis on a Beckman Coulter Multisizer and confirmed by flow cytometry phenotypic analysis. Efficient enrichment and recovery of lymphocytes from leukapheresis cell products was achieved using modified elutriation settings for flow rate and fraction volume. Elutriation allowed for enrichment of larger numbers of lymphocytes compared with depletion of monocytes by bead adherence, with a trend toward increased lymphocyte purity and yield via elutriation, resulting in a substantial reduction in the cost of enrichment per cell. Importantly, significant lymphocyte enrichment could be accomplished using leukapheresis samples from healthy donors (n=12) or from study subjects with HIV infection (n=15) or malignancy (n=12). Clinical-scale closed-system elutriation can be performed efficiently for the selective enrichment of lymphocytes for immunotherapy protocols. This represents an improvement in cost, yield and purity over current methods that require the addition of monocyte-depleting beads.Cytotherapy 01/2009; 11(7):923-35. · 3.06 Impact Factor
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ABSTRACT: The generation of Ag-loaded DC under good manufacturing practice (GMP) conditions is logistically challenging and further compounded when the starting precursors need to be purified from B-CLL patients who have overwhelming numbers of circulating B-CLL cells and decreased numbers of monocytes. We have previously demonstrated that DC with endocytosed B-CLL apoptotic bodies are powerful stimulators of anti-leukemic T cells. In this study we compared counterflow elutriation and immunomagnetic separation for enriching monocyte precursors, and evaluated the feasibility of generating DC from B-CLL patients and the effects of cryopreservation. Monocyte yield from a single leukapheresis product of a B-CLL patient varied from 1 x 108 to 10 x 108 total cells, from which 40-200 x 106 mature DC could be produced. Adequate numbers of monocytes could not be enriched from one patient with 0.2% monocytes in the leukapheresis product, and the target of 50 x 106 DC was barely achieved in another patient with 0.9% monocytes in the pheresed cells. These results suggested that successful production of DC is dependent on a minimum frequency of 1% CD14(+) monocytes in the leukapheresis product. Cryopreservation of tumor cell-loaded DC yielded a recovery rate of 86+/-4.4% upon thawing, with a total viability of 90+/-2.8%. Most importantly, cryopreserved Ag-loaded DC retained their morphology, phenotype and function. The results demonstrate that adequate numbers of functional DC required for clinical therapy can be generated from patients who have >1% of CD14(+) monocytes in the leukapheresis product. Moreover, Ag-loaded DC can be cryopreserved and recovered without significant change in phenotype or function.Cytotherapy 01/2006; 8(4):318-26. · 3.06 Impact Factor
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ABSTRACT: RNA-electroporated dendritic cell (DC)-based vaccines are rapidly gaining interest as therapeutic cancer vaccines. We report on a phase I dose-escalation trial using clinical-grade manufactured mature RNA-electroporated DC in acute myeloid leukemia (AML) patients. CD14(+) cells were isolated from leukapheresis products by immunomagnetic CliniMACS separation and differentiated into mature DC (mDC). mDC were electroporated with clinical-grade mRNA encoding the Wilm's tumor (WT1) antigen, and tested for viability, phenotype, sterility and recovery. To test product safety, increasing doses of DC were administered intradermally four times at 2-week intervals in 10 AML patients. In a pre-clinical phase, immunomagnetic monocyte isolation proved superior over plastic adherence in terms of DC purity and lymphocyte contamination. We also validated a simplified DC maturation protocol yielding a consistent phenotype, migration and allogeneic T-cell stimulatory capacity in AML patients in remission. In the clinical trial, highly purified CD14(+) cells (94.5+/-3.4%) were obtained from all patients. A monocyte-to-mDC conversion factor of 25+/-10% was reached. All DC preparations exhibited high expression of mDC markers. Despite a decreased cell recovery of mDC after a combination of mRNA electroporation and cryopreservation, successful vaccine preparations were obtained in all AML patients. DC injections were well tolerated by all patients. Our method yields a standardized, simplified and reproducible preparation of multiple doses of clinical-grade mRNA-transfected DC vaccines from a single apheresis with consistent mature phenotype, recovery, sterility and viability. Intradermal injection of such DC vaccines in AML patients is safe.Cytotherapy 07/2009; 11(5):653-68. · 3.06 Impact Factor