Cell Isolation and Expansion Using Dynabeads ®

Abstract

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.

PBMC-Derived T Cells

“…However, surface bound antibodies may elicit the transmission of signals across the cell membrane. This can be avoided by negative isolation, which does not require attachment of antibodies to the cells of interest at any time (Neurauter et al 2007 ). By negative isolation , the cells are selected by removing all other cell types from the sample.…”

“…This methodology is very effi cient in removing target cells due to its fast cell capture kinetics, whereas the use of secondary-antibody coated beads makes it a fl exible protocol to isolate any cell of interest (Neurauter et al 2007 ).…”

Cell Purification: A New Challenge for Biobanks

“…During this period, several different types of paramagnetic beads have been developed and made commercially available for cell sorting purposes. Among those most widely used are Dynabeads® (microbeads of 1-3 μm; Invitrogen, Carlsbad, Calif., USA) [59] and MACS® beads (nanobeads of 20-100 nm; Miltenyi Biotec, Bergisch Gladbach, Germany) [60]. …”

“…Cell-containing samples that can be used include PB and other single-cell suspensions derived from body fluids, cell culture or disaggregated solid tissues [59]. One of the major advantages of these beads is that they do not require any special sample preparation step apart from their incubation with the sample (including the use of a separation column or centrifugation steps).…”

Expression of mRNAs for the diacylglycerol kinase family in immune cellsduring an inflammatory reaction

“…Flow cytometry analysis was performed before and after cell isolation according to the standard protocols using FITC-conjugated antibodies (anti-CD14-FITC, anti-CD4-FITC, anti-CD8-FITC, and anti-CD19-FITC; Beckman Coulter, Brea, CA) (25).…”