Label-free purification of viable human T-lymphocyte cells from a mixture of viable and non-viable cells after transfection by electroporation

Abstract

Recent developments in chimeric antigen receptor (CAR) T-cell therapy for cancer have shown promising results. CAR T-cells are engineered mainly by AC or DC electroporation to express CAR molecules on their surfaces. Electroporation has detrimental effects on the majority of T-cells and causes cell death via apoptosis or necrosis. These non-viable cells have potentially harmful effects on other healthy T-cells. Because there are no unique biomarkers that pinpoint the apoptosis and necrosis of T-cells, currently available cell separation methods are incapable of purifying healthy T-cells after electroporation. To address this critical issue, we have used dielectrophoretic cell isolation in a simple microfluidics chip. Our results indicate that it is possible to purify cell samples after electroporation and achieve ~100% purity and >90% target cell recovery. Therefore, this work presents a viable solution to a critical need in CAR T-cell manufacturing.

Recent advances in dielectrophoresis toward biomarker detection: A summary of studies published between 2014 and 2021

“…Typically, the larger DEP force of target cells was used to deflect them from nontarget cells during the separating. In addition, in some studies, larger DEP forces on nontarget cells called negative selection were used to separate and purify cell samples [37]. In these studies, there was no need to label target or nontarget cells with fluorophore tags, required for fluorescence-activated cell separation or magnetic beads-magnetic-activated cell separation.…”

“…For this reason, dielectrophoretic cell separation was categorized as label‐free separation. However, we found that the label‐free nature of dielectrophoresis was heavily utilized in cell separation including CTC separation from blood samples [37 –39].…”

“…used DEP force to isolate live cells from a mixture of dead or dying cells suspended in 0.01× PBS [37]. This evidence shows that DEP force can be applied selectively to the molecules and cells of interest and can be used to selectively concentrate them on (using attractive DEP or pDEP) or away from (using repulsive DEP or nDEP) electrodes [37].…”

“…Moreover, pDEP of cancer cells was used to attract them on the electrodes, while at the same time, nDEP produced on PBMCs was used to repel them from electrodes. Jayasooriya et al used DEP force to isolate live cells from a mixture of dead or dying cells suspended in 0.01× PBS [37]. This evidence shows that DEP force can be applied selectively to the molecules and cells of interest and can be used to selectively concentrate them on (using attractive DEP or pDEP) or away from (using repulsive DEP or nDEP) electrodes [37].…”

Planar printed electrodes for electroporation with high EM field homogeneity

“…With over two decades of successful utilization [1][2][3][4][5][6], electroporation (EP) is a mature biotechnology and research field. It has been employed for in vitro electrotransfer of small or large molecules while avoiding viral vectors or chemical methods [7,8], ex vivo cell transfection [9], in vivo gene or drug delivery [10][11][12][13][14], inactivation of pathogens [15,16], tissue ablation by irreversible EP [17][18][19] and electrochemotherapy [20,21]. Utilization of EP in cancer therapies is particularly promising, as it targets cancer cells much more efficiently than normal cells due to different electric properties [22,23].…”