This chapter describes the use of Dynabeads for cell isolation and expansion. Dynabeads are uniform polystyrene spherical beads that have been made magnetisable and superparamagnetic, meaning they are only magnetic in a magnetic field. Due to this property, the beads can easily be resuspended when the magnetic field is removed. The invention of Dynabeads made, by Professor John Ugelstad, has revolutionized the separation of many biological materials. For example, the attachment of target-specific antibodies to the surface of the beads allows capture and isolation of intact cells directly from a complex suspension such as blood. This is all accomplished under the influence of a simple magnetic field without the need for column separation techniques or centrifugation. In general, magnetic beads coated with specific antibodies can be used either for isolation or depletion of various cell types. Positive or negative cell isolation can be performed depending on the nature of the starting sample, the cell surface markers and the downstream application in question. Positive cell isolation is the method of choice for unprocessed samples, such as whole blood, and for downstream molecular applications. Positive cell isolation can also be used for any downstream application after detachment and removal of the beads. Negative cell isolation is the method of choice when it is critical that cells of interest remain untouched, i.e., no antibodies have been bound to any cell surface markers on the cells of interest. Some cell populations can only be defined by multiple cell surface markers. Such populations of cells can be isolated by the combination of negative and positive cell isolation. By coupling Dynabeads with antibodies directed against cell surface activation molecules, the beads can be used both for isolation and expansion of the cells. Dynabeads are currently used in two major clinical applications: 1) In the Isolex 300i Magnetic Cell Selection System for CD34 Stem Cell Isolation--2) For ex vivo T cell isolation and expansion using Dynabeads ClinExVivo CD3/CD28 for clinical trials in novel adoptive immunotherapy.
"Even though magnetic beads are regarded to leave cells intact, cells without beads are more attractive for further use. Another example for such bead releasing from the cells is the Dynal system where magnetic Dynabeads are detached from the cell surface via enzymatic reaction . Especially if cells are isolated for clinical, i.e. therapeutic applications the influence of beads on the cell surface can have adverse effects and should be investigated. "
[Show abstract][Hide abstract] ABSTRACT: Isolation of different cell types from one sample by fluorescence activated cell sorting is standard but expensive and time consuming. Magnetic separation is more cost effective and faster by but requires substantial effort. An innovative pluriBead-cascade cell isolation system (pluriSelect GmbH, Leipzig, Germany) simultaneously separates two or more different cell types. It is based on antibody-mediated binding of cells to beads of different size and their isolation with sieves of different mesh-size. For the first time, we validated the pluriSelect system for simultaneous separation of CD4+- and CD8+-cells from human EDTA-blood samples. Results were compared with those obtained by magnetic activated cell sorting (MACS; two steps -first isolation of CD4+, then restaining of the residual cell suspension with anti-human CD8+ MACS antibody followed by the second isolation). pluriSelect separation was done in whole blood, MACS separation on density gradient isolated mononuclear cells. Isolated and residual cells were immunophenotyped by 7-color 9-marker panel (CD3; CD16/56; CD4; CD8; CD14; CD19; CD45; HLA-DR) using flow cytometry. Cell count, purity, yield and viability (7-AAD exclusion) were determined. There were no significant differences between both systems regarding purity (MACS (median[range]: 92.4% [91.5-94.9] vs. pluriSelect 95% [94.9-96.8])) of CD4+ cells, however CD8+ isolation showed lower purity by MACS (74.8% [67.6-77.9], pluriSelect 89.9% [89.0-95.7]). Yield was not significantly different for CD4 (MACS 58.5% [54.1-67.5], pluriSelect 67.9% [56.8-69.8]) and for CD8 (MACS 57.2% [41.3-72.0], pluriSelect 67.2% [60.0-78.5]). Viability was slightly higher with MACS for CD4+ (98.4% [97.8-99.0], pluriSelect 94.1% [92.1-95.2]) and for CD8+-cells (98.8% [98.3-99.1], pluriSelect 86.7% [84.2-89.9]). pluriSelect separation was substantially faster than MACS (1h vs. 2.5h) and no pre-enrichment steps were necessary. In conclusion, pluriSelect is a fast, simple and gentle system for efficient simultaneous separation of two and more cell subpopulation directly from whole blood and provides a simple alternative to magnetic separation.
PLoS ONE 09/2013; 8(9):e74745. DOI:10.1371/journal.pone.0074745 · 3.23 Impact Factor
"After washing, the cells were incubated with a monoclonal anti-mouse antibody directed against platelet glycoprotein IIb (CD41+; BD Biosciences, San Jose, CA) and suspended with immunomagnetic beads (Dynabeads, Invitrogen Corp., Carlsbad, CA) coated with sheep anti-mouse IgG antibody , . The CD41+ antibody has high specificity for mature and immature MKs ,  and this isolation technique yields at least 96.6% purity of MKs . "
[Show abstract][Hide abstract] ABSTRACT: Serotonin (5-HT) is a biogenic amine that also acts as a mitogen and a developmental signal early in rodent embryogenesis. Genetic and pharmacological disruption of 5-HT signaling causes various diseases and disorders via mediating central nervous system, cardiovascular system, and serious abnormalities on a growing embryo. Today, neither the effective modulators on 5-HT signaling pathways nor the genes affected by 5-HT signal are well known yet.
In an attempt to identify the genes altered by 5-HT signaling pathways, we analyzed the global gene expression via the Illumina array platform using the mouse WG-6 v2.0 Expression BeadChip containing 45,281 probe sets representing 30,854 genes in megakaryocytes isolated from mice infused with 5-HT or saline. We identified 723 differentially expressed genes of which 706 were induced and 17 were repressed by elevated plasma 5-HT.
Hierarchical gene clustering analysis was utilized to represent relations between groups and clusters. Using gene ontology mining tools and canonical pathway analyses, we identified multiple biological pathways that are regulated by 5-HT: (i) cytoskeletal remodeling, (ii) G-protein signaling, (iii) vesicular transport, and (iv) apoptosis and survival. Our data encompass the first extensive genome-wide based profiling in the progenitors of platelets in response to 5-HT elevation in vivo.
PLoS ONE 08/2013; 8(8):e72580. DOI:10.1371/journal.pone.0072580 · 3.23 Impact Factor
"Sorting methods for isolating LGCs and purifying this specific cell type are based on the negative selection of LGCs through the depletion of contaminating immune cells. Currently, employed strategies for the depletion of unwanted cells can be classified in two main groups, according to a criterion of restrictiveness: (i) highly specific strategies of depletion based on immunological recognition of specific cell markers of contaminating cells (Neurauter et al., 2007) and (ii) non-specific strategies of depletion based on the differential physical properties of contaminating cells and desired cells. (i) The methods based on the recognition of specific cell markers in contaminating immune cells are expected to be more restrictive and thus render LGCs of purity .90%. "
[Show abstract][Hide abstract] ABSTRACT: Several protocols for the isolation of luteinized granulosa cells (LGCs) contained in follicular fluid have been described but no previously published study has compared the relative efficiency of these protocols. Our objective is to obtain conclusive scientific evidence for the superiority of one method over another.
Different purification methods for LGCs based on the recognition of specific cell markers, aggregates, differential adhesion and LGC size were evaluated. We compared the levels of CD45 cell contamination and the percentage of total cell viability in paired aliquots of cells (before and after purification) derived from the follicular fluid obtained from women who were donating oocytes (n = 72). Each of the six purification methods was performed six times using pooled follicular fluids from two women.
Samples processed by means of recognition of specific cell markers were characterized by their greater purity (0.1-1.33% CD45+) but low rate of LGC recovery (17.13-25.4%) when compared with the other methods (3.29-12% CD45+, P < 0.05 and 51.67-73.20% LGC, P < 0.05). It is noteworthy that the filter method, which is based on the LGC size, combined one of the highest rates of LGC recovery (∼70%) with acceptable low levels of contamination (<5%).
There is currently no gold standard method for the isolation of LGCs, and protocols should be chosen depending on the purpose in question. We conclude that fluorescence-activated cell sorting is the best protocol for isolating LGCs when purity is the principal criterion, and magnetic separation when both purity and viability are essential. However, cell straining (filter) is probably the least laborious and, overall, the most efficient method to isolate LGCs.
Human Reproduction 03/2012; 27(6):1781-9. DOI:10.1093/humrep/des096 · 4.57 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.