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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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, cy....

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.......

Acknowledgements

The authors would like to acknowledge financial support by the National Science Foundation (NSF CBET 0967598, DBI IDBR 1353918) and the U.S. Department of Education's Graduate Training in Emerging Areas of Precision and Personalized Medicine (P200A150131) for funding graduate student J.J.S. on fellowship.

....

Materials

NameCompanyCatalog NumberComments
150-mm diameter petri dishesVWR25384-326step 6.1.1 to secure wafer
24-well tissue culture platesVWR10062-896step 10.3.6 to plate electroporated cells
33220A Waveform/Function generatorAgilentstep 9.2.3 electroporation pulse generator
4'' Si-wafersUniversity Wafersubsection 2.1 for microfluidic channel fabrication
6-well tissue culture platesVWR10062-892step 8.1.8 to plate cells
AcetoneFisher ScientificA18-4step 2.1.2 for cleaning and  step 5.1 photoresist lift-off
Allegra X-22R CentrifugeBeckman Coultersteps 8.1.4 , 8.3.2. and 8.3.3. to spin down cells
AutoCAD 2018Autodesksubsection 1.1. to design transparency masks
Buffered oxide etchant 10:1VWR901621-1Lsubsection 3.1 for HF etching
CCD Monochrome microscope cameraHamamatsuOrca 285 C4742-96-12G04step 11.2.3. for imaging
CMOS camera- Sensicam QE 1.4MPPCOsubsection 9.3 part of the experimental setup
Conductive EpoxyCircuitWorksCW2400subsection 7.6. for wire attachement
Conical Centrifuge Tubes, 15 mLFisher Scientific14-959-70Cstep 8.1.4. for cell centrifuging
Dektak 3ST Surface ProfilometerVeeco (Sloan/Dektak)step 2.1.15 and 5.4 for surface profilometry
Disposable biopsy punch, 0.75 mmRobbins InstrumentsRBP075step 6.2.3 for inlet access
Disposable biopsy punch, 3 mmRobbins InstrumentsRBP30Pstep 6.2.3 for outlet access
DRAQ5abcamab108410step 11.2.2. for live cell staining
Dulbecco’s Modified Eagle’s MediumThermoFisher Scientific11885084step 8.1.2. part of media composition
E3631A Bipolar Triple DC power supplyAgilentstep 9.2.1.-9.2.2.part of the experimental setup
Eclipse TE2000-U Inverted  MicroscopeNikon subsection 9.3. part of the experimental setup
EVG620 UV Lithography SystemEVG step 2.1.9. and 2.2.7. for UV Exposure
Fetal Bovine SerumNeuromicsFBS001step 8.1.2. part of media composition
FS20 Ultrasonic CleanerFisher Scientificsubsection 5.1. for photoresist lift-off
Glass Media Bottle with Cap, 100mLFisher ScientificFB800100step 8.2.1. for buffer storage
Glass Media Bottle with Cap, 500mLFisher ScientificFB800500step 8.1.2.for media storage
HEK-293 cell lineATCCCRL-1573subsection 8.1 for cell culturing
HEPES buffer solutionSigma Aldrich83264-100ML-Fstep 8.2.1 part of electroporation buffer composition
HexamethyldisilazaneSigma Aldrich379212-25MLstep 2.2.3 adhesion promoter
HF2LI Lock-in AmplifierZurich Instrumentssubsection 9.2 part of the experimental setup
HF2TA Current amplifierZurich Instrumentssubsection 9.2 part of the experimental setup
Isopropyl AlcoholFisher ScientificA459-1step 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 microscopeOlympusstep 11.2.3 for imaging
L-Glutamine SolutionSigma AldrichG7513-20MLstep 8.1.2. part of media composition
M16878/1BFA 22 gauge wireAWCB22-1subsection 7.5 for device fabrication
Magnesium chlorideSigma Aldrich208337-100Gstep 8.1.2 part of electroporation buffer composition
MF 319 DeveloperKayaku Advanced Materials10018042step 2.2.9. photoresist developer
Microposit S1818 photoresistKayaku Advanced Materials1136925step 2.2.4 positive photoresist for electrode patterning
Microscope slides, 75 x 25 mmVWR16004-422step 2.2.1 electrode soda lime glass substrate
Model 2350 High voltage amplifierTEGAM2350step 9.2.5. part of the experimental setup
National Instruments LabVIEWNational Instrumentsdata acquisition
Needle, 30G x 1 inBD Scientific305128step 10.1.1. part of the system priming
PA90 IC OPAMP Power circuitDigi-key598-1330-NDPart of the custom circuit
Penicillin-StreptomycinSigma AldrichP4458-20MLstep 8.1.2. part of media composition
Plasmid pMAX-GFPLonzaVCA-1003step 8.3.4. for intracellular delivery
Plastic tubing, 0.010'' x 0.030"VWR89404-300step 10.1.2. for system priming
Platinum targetsKurt J. Leskersubsection 4.2. for physical vapor deposition
Potassium chlorideSigma AldrichP9333-500Gstep 8.2.1. part of electroporation buffer composition
Pump 11 PicoPlus microfluidic syringe pumpHarvard ApparatusMA1 70-2213step 10.1.4. for system priming
PVD75 Physical vapor deposition systemKurt J. Leskersubsection 4.1. for physical vapor deposition
PWM32 Spinner SystemHeadway Researchsteps 2.1.6 and 2.2.2. for substrate coating with photoresist
PX-250 Plasma treatment systemMarch Instrumentssubsection 7.2 for PDMS and glass substrate bonding
SDG1025 Function/Waveform generatorSiglentstep 9.2.2. part of the experimental setup
Sodium hydroxideSigma AldrichS8045-500Gstep 8.2.1. part of electroporation buffer composition
SU-8 2010 negative photoresistKayaku Advanced MaterialsY111053step 2.1.7. for microfluidic channel patterning
SU-8 developerMicrochemY010200step 2.1.12. for photoresist developing
SucroseSigma AldrichS7903-1KGstep 8.2.1. part of electroporation buffer composition
Sylgard 184 elastomer kitDow Corning3097358-1004step 6.2.1  10 : 1 mixture of PDMS polymer and hardening agent
Syringe, 1 mlBD Scientific309628step 8.3.4. part of system priming
SZ61 Stereomicroscope SystemOlympussubsection 7.3. for channel and electrode alignment
Tissue Culture Treated T25 FlasksFalcon353108step 8.1.2 for cell culturing
Titanium targetsKurt J. Leskersubsection 4.2. for physical vapor deposition
Transparency masksCAD/ART Servicessteps 2.1.9. and 2.2.7. for photolithography
Trichloro(1H,1H,2H,2H-perfluorooctyl)silaneSigma Aldrich448931-10Gstep 6.1.2. for wafer silanization
Trypsin-EDTA solutionSigma AldrichT4049-100MLsteps 8.1.3. and 8.3.1. for cell harvesting

References

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