The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Summary

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

Abstract

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this ‘optimal’ pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

Introduction

Current therapeutic innovations in biomedical research, such as CAR-T (Chimeric Antigen Receptor Engineered T cell) cell therapy and genetic editing using CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9, heavily rely on the ability to deliver exogenous material both successfully and efficiently into the intracellular space¹. In CAR-T therapy, the gold standard to perform the gene delivery step in cell therapy manufacturing is using viral vectors². Though viral-mediated gene delivery is an efficient delivery modality, it also has several drawbacks. These include manufacturing costs, cytotoxicity, immunogenicity, mutagenesis/tumorigenesis potential, and size limitations on the gene(s) to be delivered³. These limitations have led to the research and development of alternative, non-viral delivery technologies.

Electroporation, an alternative to viral-mediated gene delivery, relies on the application of an optimal electrical pulse waveform to perform DNA, RNA, and protein transfections of cells. Following the application of an external electric field, the cell membrane is briefly compromised, making the cell susceptible to the intracellular delivery of otherwise impermeable exogenous materials⁴. Compared to viral-mediated delivery, electroporation is advantageous as it is generally safe, easy to operate, and has low operating costs. Electroporation can deliver both small and large molecular cargo and can be efficient in transfecting cells regardless of lineage⁵. To achieve desirable outcomes following electroporation, i.e., good viability and good electro-transfection efficiency, a variety of experimental parameters need to be co-optimized. These include cell type⁶, cell density, molecule concentration⁷, electroporation buffer properties (e.g., molecular composition, conductivity, and osmolarity)⁸, electrode size/geometry⁹, and electrical pulse waveform (shape, polarity, number of pulses)¹⁰ (refer to Figure 1 for an illustration). Although each of these parameters can have a significant effect on the outcomes of electroporation experiments, pulse waveform has been especially studied in great detail, as the electrical energy of the applied pulse(s) is the root of the intrinsic trade-off between the resulting cell viability and electro-transfection efficiency⁸.

Typically, electroporation experiments are performed on the macro-scale, where cells are suspended in 100s of microliters of buffer between a set of large, parallel-plate electrodes within an electroporation cuvette. The electrodes are commonly manufactured out of aluminum with an electrode distance of 1-4 mm. Once the cells are manually loaded via pipette, the cuvette is electrically connected to a bulky, electrical pulse generator where the user can set and apply the pulse waveform parameters to electroporate the cell suspension. Although macro-scale or bulk electroporation can process cell densities >10⁶ cells/mL, this feature can be wasteful when optimizing the electrical pulse waveform settings. This is particularly of concern when electroporating primary cell types where the cell population numbers can be limited. Additionally, due to the large distance between the electrodes, the pulse generator must be able to supply large voltages to achieve electric field strengths >1kV/cm¹¹. These high voltages cause resistive power dissipation through the electrolyte buffer resulting in Joule heating, which can be detrimental to the resulting cell viability¹². Lastly, performing electroporation on a dense suspension of cells will consistently be burdened with an innate variability in the resulting electro-transfection efficiency and cell viability. Each cell in suspension could experience a different electric field strength due to the surrounding cells. Depending on whether the experienced electric field strength is either increased or decreased, the resulting cell viability or electro-transfection efficiency may each be negatively impacted¹¹. These downsides to macro-scale electroporation have led to the pursuit and development of alternative technologies that operate on the micro-scale and allow for better control at the single-cell level.

The field of BioMEMS, or biomedical micro-electro-mechanical systems, stems from the technological advancements made in the microelectronics industry. Specifically, utilizing microfabrication processes to develop micro-devices for the advancement of biomedical research. These advancements include the development of micro-electrode arrays for in vivo electrical monitoring¹³, capacitive micro-electrodes for in situ electroporation¹⁴, miniaturized organ-on-a-chip devices¹⁵, microfluidic point-of-care diagnostics¹⁶, biosensors¹⁷, and drug delivery systems¹⁸, including nano- and micro-electroporation devices^19,20,21. Due to the ability to design and manufacture devices at the same size scale as biological cells, nano- and micro-electroporation technologies are advantageous when compared to their macro-scale counterpart^22,23. These electroporation devices eliminate the requirement of high voltage pulse applications, as electrode sets with spacings of 10s to 100s of micrometers are typically integrated. This feature drastically reduces the current through the electrolyte, which in turn reduces the accumulation of toxic electrolysis products and the effects of Joule heating in these systems. The micro-scale channels also ensure that a much more uniform electric field is reliably applied to the cells during pulse application, resulting in more consistent outcomes²⁴. In addition, it is also commonplace for micro-electroporation devices to be integrated into a microfluidic platform which lends itself for future integration into a fully automated technology, a highly desirable capability in cell therapy manufacturing²⁵. Lastly, micro-scale electroporation allows for the electrical interrogation of electroporation events. For example, the degree of cell membrane permeabilization can be monitored in real-time at a single cell level^26,27. The purpose of this method is to describe the microfabrication, system operation, and analysis of a microfluidic, single-cell micro-electroporation device capable of measuring the degree of cell membrane permeabilization for optimizing electroporation protocols, yet increasing throughput over the previous state-of-the-art.

Performing single-cell level electroporation is no longer a novel technique, as it was first demonstrated by Rubinsky et al. in 2001 with the development of a static cell electroporation technology²⁸. Their micro-device was innovative as they were the first to demonstrate the ability to electrically monitor the event of electroporation. This has further led to the development of static, single-cell electroporation technologies capable of electrically detecting the degree of cell membrane permeabilization in a parallelized manner to increase the throughputs of the devices. However, even with parallelization and batch processing, these devices severely lack the total number of cells they can process per unit time^29,30. This limitation has led to the development of flow-through devices capable of performing single-cell level micro-electroporation at much greater throughputs³¹. This device transition, from static to flow-through environment, limits the capability of electrically monitoring the degree of cell membrane permeabilization following the application of the electroporation pulse. The method described in this work bridges the gap between these two technologies, a micro-electroporation technology capable of electrically detecting, pulsing, and monitoring the degree of cell membrane permeabilization of individual cells, in a continuous-flow, serial fashion.

This technology was recently described in Zheng et al. In that work, the capabilities of this technology were introduced with the completion of a parametric study, where both the amplitude and duration of the electroporation pulse were varied, and the ensuing electrical signal, indicative of cell membrane permeabilization, was explored³². The results showed that an increase in the intensity of the electroporation pulse (i.e., increase in applied electric field or increase in pulse duration) caused an increase in the measured cell membrane permeabilization. To further validate the system, a common fluorescent indicator of successful electroporation, propidium iodide³³, was added to the cell suspension, and a fluorescence image was captured immediately following the application of the electrical pulse. The optical signal, i.e., the fluorescence intensity of propidium iodide inside the cell, was strongly correlated with the electrical measurement of the degree of cell membrane permeabilization, verifying the reliability of this electrical measurement. However, this work only considered the delivery of the small molecule propidium iodide, which has little to no translatable significance.

In this work, a new application of this technology is introduced to improve upon the throughput of the system while delivering a biologically active plasmid DNA (pDNA) vector and assessing the electro-transfection efficiency of cells replated and cultured following electroporation. Though the previous work outperforms existing micro-electroporation technologies that are capable of electrically measuring the event of electroporation, the current state of the device still requires long cell transit times between the electrode set (~250 ms) to perform the cell detection, pulse application, and the cell membrane permeabilization measurement. With a single channel, this limits the throughput to 4 cells/s. To combat this limitation, a new concept of cell-population-based feedback-controlled electroporation is introduced to perform pDNA electro-transfection. By using a hypo-physiologic conductivity electroporation buffer, this system allows for the electrical interrogation of single cells across a multitude of electroporation pulse applications. Based on the electrical response, an 'optimal' electroporation pulse is then determined. A 'high-throughput' mode is then implemented where the cell membrane permeabilization determination is nullified, the flow rate is increased, and the electroporation pulse duty cycle is matched to the cell transit time to ensure one pulse per cell in transit between the electrodes. This work will provide extensive details into the microfabrication steps for the manufacturing of the micro-device, the material/equipment and their setup required to perform the experimentation, and the operation/analysis of the device and its electro-transfection efficiency (eTE).

Figure 1: Experimental factors affecting electroporation outcomes. (Left) Cell Suspension-Important factors to consider prior to the onset of electroporation include: Payload (in this case, pDNA), concentration, cell density, and electroporation buffer properties. Electroporation buffer properties to consider are conductivity, osmolarity, and the exact molecular composition contributing to these values. (Middle) Pulse Application-The exact pulse-type (square wave vs. exponential decay) and pulse waveform (single pulse vs. pulse train) must be optimized to maximize both the resulting cell viability and electro-transfection efficiency. Common pulse trains implemented in electroporation processes are typically composed of a series of High Voltage (HV) pulses or series of pulses rotating between HV and Low Voltage (LV) pulse magnitudes. (Right) Cell Recovery-Down-stream processing steps, in particular, the recovery cell culture media that cells are transferred to, should be optimized. Not featured (Far Left), additional upstream cell processing steps can be implemented for overall electroporation process optimization. Please click here to view a larger version of this figure.

Protocol

NOTE: Users should review all MSDS for the materials and supplies used in this protocol. Appropriate PPE should be worn at each step and sterile technique used during experimentation. Sections 1-7 discuss the device fabrication. 1. Device fabrication- Mask design NOTE: Refer to Figure 2 for an illustration of the microfabrication process. The microfabrication steps are to be carried out in a cleanroom environment. Additio…

Representative Results

Figure 4 highlights the operating principles behind the single-cell-level membrane permeabilization detection for a single pulse amplitude. Following the initiation of the electroporation experiment, the cell detection algorithm determines an optimal threshold for cell detection via a point-by-point, slope-based detection method. The system then continuously monitors (1) for a significant negative change in the measured electrical current, which is indicative of the entry of a cell. This is …

Discussion

The methodology presented within this protocol primarily focuses on the microfabrication of a microfluidic device that is then integrated into a specialized electroporation experimental setup. The term 'recipe', which is often used when describing the specifics of the microfabrication process, hints at the importance of following/optimizing each step to successfully fabricate a functioning device. However, certain critical steps within the process, when not optimized, such as UV exposure time/energy, PVD sputteri…

Materials

150-mm diameter petri dishes	VWR	25384-326	step 6.1.1 to secure wafer
24-well tissue culture plates	VWR	10062-896	step 10.3.6 to plate electroporated cells
33220A Waveform/Function generator	Agilent		step 9.2.3 electroporation pulse generator
4'' Si-wafers	University Wafer		subsection 2.1 for microfluidic channel fabrication
6-well tissue culture plates	VWR	10062-892	step 8.1.8 to plate cells
Acetone	Fisher Scientific	A18-4	step 2.1.2 for cleaning and  step 5.1 photoresist lift-off
Allegra X-22R Centrifuge	Beckman Coulter		steps 8.1.4 , 8.3.2. and 8.3.3. to spin down cells
AutoCAD 2018	Autodesk		subsection 1.1. to design transparency masks
Buffered oxide etchant 10:1	VWR	901621-1L	subsection 3.1 for HF etching
CCD Monochrome microscope camera	Hamamatsu	Orca 285 C4742-96-12G04	step 11.2.3. for imaging
CMOS camera- Sensicam QE 1.4MP	PCO		subsection 9.3 part of the experimental setup
Conductive Epoxy	CircuitWorks	CW2400	subsection 7.6. for wire attachement
Conical Centrifuge Tubes, 15 mL	Fisher Scientific	14-959-70C	step 8.1.4. for cell centrifuging
Dektak 3ST Surface Profilometer	Veeco (Sloan/Dektak)		step 2.1.15 and 5.4 for surface profilometry
Disposable biopsy punch, 0.75 mm	Robbins Instruments	RBP075	step 6.2.3 for inlet access
Disposable biopsy punch, 3 mm	Robbins Instruments	RBP30P	step 6.2.3 for outlet access
DRAQ5	abcam	ab108410	step 11.2.2. for live cell staining
Dulbecco’s Modified Eagle’s Medium	ThermoFisher Scientific	11885084	step 8.1.2. part of media composition
E3631A Bipolar Triple DC power supply	Agilent		step 9.2.1.-9.2.2.part of the experimental setup
Eclipse TE2000-U Inverted  Microscope	Nikon		subsection 9.3. part of the experimental setup
EVG620 UV Lithography System	EVG		step 2.1.9. and 2.2.7. for UV Exposure
Fetal Bovine Serum	Neuromics	FBS001	step 8.1.2. part of media composition
FS20 Ultrasonic Cleaner	Fisher Scientific		subsection 5.1. for photoresist lift-off
Glass Media Bottle with Cap, 100mL	Fisher Scientific	FB800100	step 8.2.1. for buffer storage
Glass Media Bottle with Cap, 500mL	Fisher Scientific	FB800500	step 8.1.2.for media storage
HEK-293 cell line	ATCC	CRL-1573	subsection 8.1 for cell culturing
HEPES buffer solution	Sigma Aldrich	83264-100ML-F	step 8.2.1 part of electroporation buffer composition
Hexamethyldisilazane	Sigma Aldrich	379212-25ML	step 2.2.3 adhesion promoter
HF2LI Lock-in Amplifier	Zurich Instruments		subsection 9.2 part of the experimental setup
HF2TA Current amplifier	Zurich Instruments		subsection 9.2 part of the experimental setup
Isopropyl Alcohol	Fisher Scientific	A459-1	step 2.1.2 for cleaning, step 2.1.14 for rinsing wafer following SU-8 development, and step 6.3.1 for cleaning PDMS
IX81 fluorescence microscope	Olympus		step 11.2.3 for imaging
L-Glutamine Solution	Sigma Aldrich	G7513-20ML	step 8.1.2. part of media composition
M16878/1BFA 22 gauge wire	AWC	B22-1	subsection 7.5 for device fabrication
Magnesium chloride	Sigma Aldrich	208337-100G	step 8.1.2 part of electroporation buffer composition
MF 319 Developer	Kayaku Advanced Materials	10018042	step 2.2.9. photoresist developer
Microposit S1818 photoresist	Kayaku Advanced Materials	1136925	step 2.2.4 positive photoresist for electrode patterning
Microscope slides, 75 x 25 mm	VWR	16004-422	step 2.2.1 electrode soda lime glass substrate
Model 2350 High voltage amplifier	TEGAM	2350	step 9.2.5. part of the experimental setup
National Instruments LabVIEW	National Instruments		data acquisition
Needle, 30G x 1 in	BD Scientific	305128	step 10.1.1. part of the system priming
PA90 IC OPAMP Power circuit	Digi-key	598-1330-ND	Part of the custom circuit
Penicillin-Streptomycin	Sigma Aldrich	P4458-20ML	step 8.1.2. part of media composition
Plasmid pMAX-GFP	Lonza	VCA-1003	step 8.3.4. for intracellular delivery
Plastic tubing, 0.010'' x 0.030"	VWR	89404-300	step 10.1.2. for system priming
Platinum targets	Kurt J. Lesker		subsection 4.2. for physical vapor deposition
Potassium chloride	Sigma Aldrich	P9333-500G	step 8.2.1. part of electroporation buffer composition
Pump 11 PicoPlus microfluidic syringe pump	Harvard Apparatus	MA1 70-2213	step 10.1.4. for system priming
PVD75 Physical vapor deposition system	Kurt J. Lesker		subsection 4.1. for physical vapor deposition
PWM32 Spinner System	Headway Research		steps 2.1.6 and 2.2.2. for substrate coating with photoresist
PX-250 Plasma treatment system	March Instruments		subsection 7.2 for PDMS and glass substrate bonding
SDG1025 Function/Waveform generator	Siglent		step 9.2.2. part of the experimental setup
Sodium hydroxide	Sigma Aldrich	S8045-500G	step 8.2.1. part of electroporation buffer composition
SU-8 2010 negative photoresist	Kayaku Advanced Materials	Y111053	step 2.1.7. for microfluidic channel patterning
SU-8 developer	Microchem	Y010200	step 2.1.12. for photoresist developing
Sucrose	Sigma Aldrich	S7903-1KG	step 8.2.1. part of electroporation buffer composition
Sylgard 184 elastomer kit	Dow Corning	3097358-1004	step 6.2.1  10 : 1 mixture of PDMS polymer and hardening agent
Syringe, 1 ml	BD Scientific	309628	step 8.3.4. part of system priming
SZ61 Stereomicroscope System	Olympus		subsection 7.3. for channel and electrode alignment
Tissue Culture Treated T25 Flasks	Falcon	353108	step 8.1.2 for cell culturing
Titanium targets	Kurt J. Lesker		subsection 4.2. for physical vapor deposition
Transparency masks	CAD/ART Services		steps 2.1.9. and 2.2.7. for photolithography
Trichloro(1H,1H,2H,2H-perfluorooctyl)silane	Sigma Aldrich	448931-10G	step 6.1.2. for wafer silanization
Trypsin-EDTA solution	Sigma Aldrich	T4049-100ML	steps 8.1.3. and 8.3.1. for cell harvesting