Long‐term in vitro persistence of magnetic properties after magnetic bead‐based cell separation of T cells

Abstract

Magnetic‐activated cell sorting (MACS) using magnetic nanoparticles coated with specific antibodies is commonly used in immunology research. For in vitro isolation purposes, it is important to know to what extent the magnetic properties remain present in the isolated cell populations and whether it has consequences for sequential isolations. We hypothesized that only upon cell division, cells will lose their magnetic properties via dilution of the particles in/on their daughter cells. We analysed residual magnetic properties of cells that divided vs cells that did not divide after magnetic bead‐based cell separation. As a model, we isolated T cells using beads targeting the non‐modulating surface molecule CD45RO. Cells were labelled with the cell division tracking dye PKH and cultured under different conditions to induce variable degrees of cell division. We demonstrate that T cells that underwent no, or only minimal, cell divisions after MACS retained magnetic properties for up to at least 2 weeks of in vitro culture. The presence of nanoparticles was detected on their cell surface and intracellularly using Labeling Check reagent. These results have important consequences for procedures requiring repetitive isolation rounds after in vitro culture.

Label-free lymphocytes reconstitution using side scatter for optimal T cell manufacturing

“…Superparamagnetic particles-antibody coupling technique combined with magnetic isolation is shown to be capable of minimizing the effects introduced by fluorescent antibodies occupancy thanks to its effective separation with only unsaturated concentration of bead-antibody conjugates (Grutzkau and Radbruch, 2010; Plouffe et al, 2015). However, concerns arising from the use of nanosized magnetic beads include internalization of magnetic beads and preservation of magnetic properties for an extended period in cells that are subjected to limited proliferation, which calls for caution when repetitive positive isolation steps after in vitro culture are required (Laghmouchi et al, 2020; Teeman et al, 2019). More recently, Miltenyi Biotec has introduced the REAlease Fluorochrome Technology that relies on engineered antibody fragments with low epitope binding affinities which allows stained antibodies to be released.…”

Isolation and characterization of peritoneal microvascular pericytes

“…The growth of pericytes also inhibited the proliferation of other ingredients when the culture conditions changed, which was confirmed in the present study by flow cytometry. Fluorescence‐activated cell sorting, differential centrifugation and MACS are common methods for cell separation [ 21 ]. Fluorescence‐activated cell sorting is time‐consuming and easily contaminated, and the survival rate of cultured cells is low.…”

Proof-of-Concept of a Novel Cell Separation Technology Using Magnetic Agarose-Based Beads

“…Magnetic particles have to be biocompatible, as any toxic effects might compromise the cell isolation as well as the cell quality and the therapeutic efficiency of the cell-based treatment [26]. The MAGicBeads consist of biodegradable material, such as agarose and iron oxide, and these materials are known to be not harmful to cells [27]. To ensure the MAGicBeads biocompatibility for stem cells, a toxicity test was performed with hMSCs.…”

“…Furthermore, possible multiple attachment points for the cells can provide a more robust system for a potential scale-up. Additionally, studies have shown that the cells can retain the magnetic properties of the IMS up to 2 weeks after initial isolation [27] providing a potential safety risk. In the same study, it was also shown that these small magnetic beads can be taken up by the cells and have been found on the cell surface and in the intra-cellular spaces [27].…”