High-Throughput Capable Three-Dimensional Tissue Model for Quantification of Electroporation Thresholds

Summary

This protocol uses computational modeling to quantify reversible and irreversible electroporation thresholds using the spatial distribution of transfected cells within a three-dimensional tissue mimic for high-throughput analysis of electroporation protocols.

Abstract

Electroporation is a promising technology utilizing electrical pulses for macromolecule delivery and soft-tissue ablation, with applications that include next-generation prophylactics and the treatment of genetic diseases such as cancer. This study demonstrates a high-throughput capable 3D tissue culture model for quantification of the reversible and irreversible electroporation thresholds for a given electroporation protocol. By using a non-uniform electric field and analyzing the spatial distribution of transfected cells, both reversible and irreversible thresholds can be identified within a single sample, increasing the efficiency at which electroporation protocols can be characterized, especially for in vivo translation. To show this capability, 3D tissue mimics containing HEK293 cells were transfected using a ring and pin electrode to deliver a GFP-encoding plasmid. Electroporation thresholds were then derived based on fluorescent microscopy images of the transfected samples. This model demonstrates potential for use as a means for high-throughput evaluation of electroporation protocols, a key advantage over current methods to evaluate these thresholds, which tend to be time-intensive and are less representative of in vivo conditions.

Introduction

Reversible electroporation (RE) is a well-established methodology for delivering a variety of normally membrane-impermeable compounds and molecules into cells^1,2,3. Here, pulsed electric fields (PEFs) are used to achieve a critical electric field intensity that induces transient, localized nanopores along portions of the cell membrane. RE has been used to deliver a variety of molecules and compounds ranging from dyes and nucleic acids to chemotherapeutics and other drugs^4,5,6,

Protocol

The reagents and the equipment used in this study are listed in the Table of Materials.

1. Cell preparation

1. In a biosafety cabinet, plate 1 × 10⁵ cells/mL of cell line HEK293 in a volume of 10 mL per tissue culture T75 flask in Eagle's Minimum Essential Medium supplemented with 10% (v/v) fetal bovine serum and 1% penicillin-streptomycin.
2. Incubate HEK293 cells in a humidified incubator at 37 °C with 5% CO₂ environment.
3. Harvest cells for experiments 2 days after seeding by removing the culture media and treating cells with 4 mL of 0.25% tryps....

Results

The ring and pin electrode successfully delivered a DNA plasmid encoding a green fluorescent protein (GFP) into 3D collagen tissue mimics embedded with HEK293 cells. Successful RE threshold quantification was achieved by measuring the outer radius of the transfected region and deriving the corresponding field intensity from the computational model of the experimental setup, as seen in Figure 2. The IRE threshold was similarly quantified by measuring the inner.......

Discussion

With this method, reversible and irreversible electroporation thresholds can be identified without the need to test multiple samples at discrete voltages. The use of a non-uniform electric field enables quantification based on the spatial distribution of transfected cells in the model. However, there are limitations associated with this non-uniform distribution. Foremost, the RE and IRE thresholds described herein are the thresholds for successful transfection and cell death, respectively, which may not be direct measure.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% trypsin-EDTA	Gibco	25200056
1000 uL pipette tips	Fisher Scientific	13-811-164
15 mL conical tube	Fisher Scientific	14-959-70C
200 uL pipette tips	Fisher Scientific	13-811-139
304 stainless steel blunt tip needles	McMaster Carr	75165A753
316 Stainless steel tubing	McMaster Carr	89785K319
Acrylic	McMaster Carr	8505K754
Biosafety Cabinet	Fisher Scientific	13-261-312
CAD software	Dassault Systems	SolidWorks
Centrifuge	Nuaire	NU-C200R
Electroporator	Hbio	45-0662	A custom built device and a BTX ECM 830 system were used in this study
EMEM	Fisher Scientific	MT10009CV	This may vary  if using a cell line other than HEK293
Falcon 12-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate, with Lid	Corning	353043	Any 12 well plate can be used, just ensure that well dimensions are taken into consideration when fabricating the electrode holder.
FBS	Fisher Scientific	A5670701
Fiber Optic Temperature Probe	Micronor Inc.	TS5-20MM-02
GFP-Plasmid	Aldeveron	gWiz-GFP
HEK293 Cells	ATCC	CRL-1573	Other cell lines can be used, just ensure the correct culture medium is used
Hot Plate	Thermoelectric Cooling America Corporation	AHP-301CPV
Ice or Refrigerated Bead Bath	Fisher Scientific	10-876-001
Image analysis software	Leica	LAS X
Incubator	Fisher Scientific	15-015-2633
Inverted Fluorescence Microscope	Leica	DMi8
Laser Cutter	McMaster Carr	7344N11
Penicillin-streptomycin	Fisher Scientific	15140122
Pipette Kit	Fisher Scientific	14-388-100
Simulation Software	COMSOL	COMSOL Multiphysics 6.2
T75 Flask	Fisher Scientific	156499
Type I Bovine Collagen Solution	Advanced Biomatrix	5005	3 mg/mL