Abstract
Background aims
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.
Methods
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.
Results
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).
Conclusions
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.
Acknowledgments
The authors would like to thank Tim Taga and Linda Taylor from CaridianBCT Inc. for providing insightful suggestions, commentary and discussions, and Lori Landgrebe, Dawn Maier, Alexander Malykhin, Linda Cheung, Ashley Vogel and Anne Chew for technical assistance. Supported by the Juvenile Diabetes Research Foundation Collaborative Center for Cell Therapy, the Leukemia and Lymphoma Society of America UPENN/CHOP Center for Experimental Biologic Therapiesand NIAID UO1 AI066290-01, NCI R21CA119538.
Statement on potential conflicts of interest: There was no financial support provided by CaridianBCT to the investigators or the laboratory beyond the customary training and technical support offered when purchasing a new instrument. Selection and analysis of data and composition of the manuscript was performed solely by the authors.