Magselectofection: an integrated method of nanomagnetic separation and genetic modification of target cells

Abstract

Research applications and cell therapies involving genetically modified cells require reliable, standardized, and costeffective methods for cell manipulation. We report a novel nanomagnetic method for integrated cell separation and gene delivery. Gene vectors associated with magnetic nanoparticles are used to transfect/transduce target cells while being passaged and separated through a high gradient magnetic field cell separation column. The integrated method yields excellent target cell purity and recovery. Nonviral and lentiviral magselectofection is efficient and highly specific for the target cell population as demonstrated with a K562/Jurkat T-cell mixture. Both mouse and human enriched hematopoietic stem cell pools were effectively transduced by lentiviral magselectofection, which did not affect the hematopoietic progenitor cell number determined by in vitro colony assays. Highly effective reconstitution of T and B lymphocytes was achieved by magselectofected murine wild-type lineage-negative Sca-1 ؉ cells transplanted into Il2rg ؊/؊ mice, stably expressing GFP in erythroid, myeloid, T-, and B-cell lineages. Furthermore, nonviral, lentiviral, and adenoviral magselectofection yielded high transfection/ transduction efficiency in human umbilical cord mesenchymal stem cells and was fully compatible with their differentiation potential. Upscaling to a clinically approved automated cell separation device was feasible. Hence, once optimized, validated, and approved, the method may greatly facilitate the generation of genetically engineered cells for cell therapies. (Blood. 2011;117(16): e171-e181) IntroductionThe feasibility of using genetically engineered cells for therapy in humans has been demonstrated using various cell types, including tumor cells, 1-3 lymphocytes, dendritic cells, 4,5 fibroblasts, 6,7 hematopoietic stem cells (HSCs), 8,9 and mesenchymal stem cells (MSCs). 10,11 Actual or potential applications are as diverse as immune gene therapy for the treatment of cancer, 1-3 cancer therapy with T cells expressing chimeric T-cell receptors, 12,13 the treatment of hereditary diseases, 8,9,[14][15][16] and a plethora of applications in regenerative medicine. 17 With the emerging field of induced pluripotent stem cells, research in genetically engineered cell therapies has reached yet another level of pace and dimension. Clinical applications will require efficient, reliable, standardized methods for cell manipulation. For optimized reproducibility and wide practicality in a decentralized manner, such methods should compose a minimum number of handling steps and be cost-effective and amenable to automation in a closed system.The goal of this work is to provide a novel methodology for performing genetic modification and cell isolation in a single standardized procedure, which we call "magselectofection" ( Figure 1). It integrates nanomagnetic cell separation, which is an approved clinical application, 18,19 and nanomagnetically guided nucleic acid delivery known as magnetofection. [20][21][22] For magneti...

Nanomagnetic Activation as a Way to Control the Efficacy of Nucleic Acid Delivery

“…For NIH 3T3 mouse fibroblasts, a 2-fold increase in reporter expression was observed. In another study, non-specific magnetic labeling of human umbilical cord mesenchymal stem cells with magnetic nanoparticles with 50 pg applied iron per cell resulted in a 2-fold increase in transduction efficiency compared with cells labeled with CD105 MicroBeads immediately before magnetofection (14). In this study, magnetic lentiviral complexes were used, and magnetotransduction was performed using a cell separation column as a magnetic device.…”

“…In this case, the internalized magnetic particles may enhance local field gradients under applied magnetic fields and serve as a "magnetic seed" to concentrate the vector at the cell surface (15). In the novel Magselectofection technology reported recently (14), a magnetic separation column is used as a magnetic device for magnetofection to associate magnetic delivery vectors with specifically labeled suspended cells directly in the column. This method resulted in further improvement of the results compared with the classical magnetofection procedure.…”

“…Non-specific magnetic cell labeling can increase the efficacy of subsequent transfection or transduction with magnetic delivery vectors (13,14). In this case, the internalized magnetic particles may enhance local field gradients under applied magnetic fields and serve as a "magnetic seed" to concentrate the vector at the cell surface (15).…”

Gene activated matrices for bone and cartilage regeneration in arthritis

“…The thermo-sensitive polymer will serve as a scaffold for MSCs, and spatiotemporal control of growth factor release will be achieved with genes triggered by exo genous signals (temperature, doxycycline) to direct cartilage and subchondral bone regeneration (49,50) . Alternatively, MSC can be transduced ex vivo by magnetofection (51,52) and then injected within the thermo-sensitive polymer into the damaged cartilage and activated as above.…”

“…Alternatively an approach using magnetically enhanced adenoviral gene transfer to human MSC in monolayer culture has been successfully established using AdenoMag and the Viro-MICST infection method on a magnetic cell separation column (51,52) .…”