Isolation of monocytes from human peripheral blood using immuno‐affinity expanded‐bed adsorption

Abstract

A novel technique for the separation of monocytes from human peripheral blood preparations has been developed. The technique is based on the use of expanded-bed adsorption and a solid perfluorocarbon derivatized with avidin or streptavidin for the indirect positive or negative capture of cells labeled with biotinylated monoclonal antibodies. The perfluorocarbon support was prepared and characterized and the contactor design and operating conditions, that enable cells to be selectively isolated, were investigated. Experiments consisted of applying an immunolabeled pulse of 1 x 10(8) peripheral blood mononuclear cells (PBMCs), isolated by density gradient centrifugation, directly onto a refrigerated expanded bed. The major cell types remaining were T-lymphocytes, B-lymphocytes, and monocytes. Monocytes could be positively adsorbed, following labeling with anti-CD14 mAb, with a clearance of up to 89% and a depletion factor of 7.6. They could also be "eluted" using mechanical shear, with a 77% yield of the applied cells at a purity of 90% and >/= 65% viability. Negative isolation of monocytes, following labeling of the other cells present with anti-CD2, CD7, CD16, CD19, and CD56 mAbs, resulted in lymphocyte depletions of up to 81% with a monocyte enrichment factor of 3.8 and purity of 71%. The monocyte viability in the flowthrough was assessed to be > 95%. This combination of expanded-bed adsorption and fluidizable affinity supports shows significant potential for the intensification of cell separations.

Enrichment using antibody‐coated microfluidic chambers in shear flow: Model mixtures of human lymphocytes

“…Yields and purity can be comparable to FACS and MACS with reasonable throughput (10 8 -10 9 cells/h) (Putnam et al, 2003). To increase active surface area per unit volume, most macroscopic CAC systems adopt a packed bed design, giving rise to residence times in the order of 1-2 h (Putnam et al, 2003;Ujam et al, 2003). Long residence time was also required in a hollow fiber design (Mandrusov et al, 1995).…”

Efficient elusion of viable adhesive cells from a microfluidic system by air foam

“…Potential approaches to release captured cells from affinity-based microfluidic devices include chemical methods such as gradient elution and mechanical methods such as the application of flow shear stress [9][10][11][12][13] and air bubbles. 14,15 For gradient elution methods, although a proteolytic enzyme combined with surfactant can release the captured cells, the surfactant would potentially disrupt the cell membrane and cause the degradation of surface markers, thus limiting the downstream biological analysis of cells.…”

“…17 For flow-induced cell detachment, studies found in open literature show that the recovery efficiency of the captured cells ranges from 60% to 80%. [10][11][12] Cheung et al 9 investigated the characteristics of the detachment of the isolated cells under flow acceleration in a bio-functionalized micro-channel; in this study, detachment efficiency of $90% of bounded cells required a high shear stress $200 dyn/cm 2 , which significantly decreased cell viability and caused changes in gene expression. 3,18 Using bubble-induced detachment of affinity-adsorbed cells has been reported in recent years.…”

Expanded bed adsorption/desorption of proteins with Streamline Direct CST I adsorbent

“…The technology of an expanded bed adsorption (EBA) has been widely applied to capture proteins directly from crude unclarified feedstocks, such as, E. coli homogenate, yeast, fermentation, mammalian cell culture, milk, animal tissue extracts (Anspach et al, 1999;Bai and Glatz, 2003;Chase, 1994;Chen et al, 2003;Clemmitt and Chase, 2002;Hjorth, 1997;Smith et al, 2002;Thommes et al, 1995;Ujam et al, 2003). The objectives of EBA process are both the removal of the bulk impurities, such as cells, cell debris, particulate matter, and contaminants, and the concentration and stabilization of the target protein molecules from the source materials.…”