Flow‐Through Electroporation of HL‐60 White Blood Cell Suspensions using Nanoporous Membrane Electrodes

Abstract

A flow-through electroporation system, based on a novel nanoporous membrane/electrode design, for the delivery of cell wall-impermeant molecules into model leukocytes, HL-60 promyelocytes, was demonstrated. The ability to apply low voltages to cell populations, with nm-scale concentrated electric field in a periodic array, contributes to high cell viability. With applied biases of 1-4V, delivery of target molecules was achieved with 90% viability and up to 65% transfection efficiency. More importantly, the system allowed electrophoretic pumping of molecules from a microscale reservoir across the membrane/electrode system into a microfluidic flow channel for transfection of cells, a design that can reduce reagent amount by eightfold compared to current strategies. The flow-through system, which forces intimate membrane/electrode contact by using a 10μm channel height, can be easily scaled-up by adjusting the microfluidic channel geometry and/or the applied voltage pulse frequency to control cell residence times at the cell membrane/electrode interface. The demonstrated system shows promise in clinical applications where low-cost, high cell viability and high volume transfection methods are needed without the risk of viral vectors. In particular genetic modification of freely mobile white blood cells to either target disease cells or to express desired protein/enzyme biomolecules is an important target platform enabled by this device system.

High Throughput and Highly Controllable Methods for In Vitro Intracellular Delivery

“…Porous substrate electroporation is a particularly promising and under investigated engineered substrate method which we reviewed in detail. [155] -2000 µg mL −1 [154] DMEM, [154] PBS [155] [ 154,155] PI 0.668 0.6 nm r h + 2 [153] -20 [152] µg mL −1 , 100 µM [167] PBS [153] , high glucose DMEM [167] [ 152,153,159,160,163,165,167] Oligonucleotide Hypo-osmolar buffer, [152] Iso-osmolar buffer, [152] PBS [158] [ Plasmid DNA CS1-CAR 9 kb - [165] GFP ≈2000 3.3 kb -0.2 [151] -1000 [153] µg mL −1 DMEM [153] [ 151,153,159] gWiz GFP ≈3500 5.8 kb -<50 µg mL −1 [156] gWiz SEAP ≈4000 6.6 kb -5 [167] -100 [157] µg mL −1 High glucose DMEM [157,167] [ 156,157,167] mCherry ≈2400 4 kb -20 µg mL −1 [152] NF2 CRISPR/ Cas9 KO >5500 >9 kb - [168] OSKM pCAG ≈7900 13 kb - [160] pDsRed-C1 ≈2900 4.7 kb -100 µg mL −1 [154] pmaxGFP ≈2100 3.5 kb -5 µg mL −1 [167] High Glucose DMEM [167] [ 160,<...>…”

“…The last category of high throughput, high control systems, porous substrate systems ( Figure 2H) are substrates containing numerous micro-and nanopores on which cells are seeded and electroporated. Porous substrates can consist of commercially available membranes with random pore distribution, [151][152][153][154][155][156][157][158][159] or uniform arrays of pores on silicon chips. [160][161][162][163][164][165][166][167][168][169][170] Like nanostructures, cells seal around the pores which limits the electric field exposure to discrete regions of each cell.…”

“…[154,156] Porous membrane systems primarily utilize track-etched polymer membranes, which have been used commercially for filtration purposes, although anodic alumina membranes have also been used. [154,155,157] Moreover, track etching is a simpler process than multiple deep reactive ion etching (DRIE) and photolithographical steps, and the membranes required for these systems are already commercially available, making porous membrane systems easier to scale up for medical and industrial applications. In addition to their availability, porous membranes are much softer and more elastic than silicon, making them more physiologically relevant than silicon which has a stiffness of over 100 GPa, [183] much higher than the 20 GPa stiffness of cortical bone.…”

“…Pulse frequencies have been reported from 1-200 Hz. The number of pulses applied per train range from 1-2400 pulses, while the number of trains is generally one and HeLa cervical epithelial Poly-L-Lysine [151] or Fibronectin [151,154] >95% [151] >80% [151] [ 151,153,154,159] HL-60 Promyeloblast, suspended >90% 65% a) [155] HT1080 Connective tissue Fibronectin [152] 50% [153] [ 152,153,159] Jurkat T lymphocyte, suspended Poly-L-Lysine or Fibronectin, [151] PEG-Silane [163] >95% [151] 75% [151] [ 151,163] KG1a Promyeloblast, suspended PEG-Silane 96% [163] K562 Lymphoblast, suspended PEG-Silane [163] 92% [163] 83.4% [163] [ 163,164] MDA-MB231 Mammary epithelial Fibronectin >99% [152] 70% [152] [ 152,158] NK-92 Natural killer, suspended 90% 74% [165] Mouse NIH3T3 Embryonic fibroblast Poly-l-lysine or fibronectin [151] >95% [151] 75% [151] […”

“…Reproduced with permission. [ 155 ] Copyright 2016, Wiley‐VCH. ii) polycarbonate membrane microfluidic device (scale bar = 12 mm).…”