An Electroporation Cytometry Protocol for Live-Cell, Fluorescence Microscopy Using U2 OS Cell Culture

Summary

Electroporation is the use of pulsed electric fields (PEFs) to create transient pores in cell cultures to introduce molecular cargo. This technique has recently become widely used in research and clinical settings. Here, we describe electroporation techniques using the recently developed "Electroporation Cytometry System" for performing live-cell, fluorescence microscopy.

Abstract

Pulsed electric fields (PEFs) have a wide range of applications in medical research and clinical applications. A key area of research focuses on electroporation (reversible or irreversible). Reversible electroporation has been used for several decades for transferring molecules through cell membranes such as plasmid DNA, typically referred to as gene electrotransfer (GET). Conversely, irreversible electroporation has become a popular technique in cancer treatment, providing a non-toxic alternative by permanently rupturing cells using PEFs. This can be combined with various types of drugs or reagents to enhance the effect which has become known as electrochemotherapy (ECT). However, despite the broad success in practical applications, further supporting technology and research is required to improve current techniques. To address this, an electroporation cytometry system was developed to support live-cell PEF experimentation with the ability to perform long-term fluorescence microscopy. This was in combination with a stably expressing FUCCI(CA)5 U2-OS cell line for assessing changes in the cell cycle in response to PEF exposure. In doing so, up to 30 h timelapse microscopy was achieved, providing real-time changes in cell activity based on the FUCCI(CA)5 reporter. As a result, phase-specific changes were quantifiable for downstream analysis.

Introduction

Pulsed electric fields (PEFs) have grown to become a common tool for a variety of applications in biology and clinical applications¹. These can be broadly categorized by electroporation (EP) and electrostimulation (ES). Electroporation has two subfields known as reversible and irreversible electroporation. Reversible electroporation (RE) has been used for decades as an effective tool for transferring molecular cargo such as plasmid DNA into cells of various types such as mammalian, bacterial, and yeast². More recently, irreversible electroporation (IRE) has grown in prominence as a non-toxic alternative to current cancer treatments³. An emerging subfield is referred to as Pulsed Field Ablation (PFA), which focuses on non-thermal tissue removal, in particular for treating certain cardiac dysfunctions⁴. In combination with drugs and other reagents, this has led to the development of electrochemotherapy (ECT). However, the current understanding of PEF treatments is being outpaced by clinical applications⁵. Here we present an electroporation cytometry system for investigating electrochemotherapies. The cell cycle represents a key indicator of cell viability, and changes can identify phase-specific effects. By developing these tools, protocols can be adapted faster, providing more effective treatments for a wider range of cancers.

Our custom electrocytometry system (Figure 1) comprises a chamber with aluminum parallel plate electrodes and a coverglass bottom as well as a simple exponential decay pulse generator. The chamber is constructed from a 3D-printed chamber and a #1.5 cover glass bottom is affixed by medical-grade pressure-sensitive adhesive. Electrodes are formed from adhesive aluminum film placed over supports built into the chamber, providing two parallel electrodes 0.4 cm apart. This is a common configuration for commercial electrodes and provides a robust design that could quickly adapt current protocols. The chamber can fit within environmental enclosures designed for 25 mm x 55 mm slides, which is also a common slide format supported by commercial microscope providers. The pulse generator provides up to 330 V exponential decay pulses, allowing for a wide range of experiments to be achieved, including reversible and irreversible electroporation.

To study the effects of PEFs on the cell cycle, we used a transgenic human osteosarcoma cell line (U2 OS) expressing the Fluorescent Ubiquitination Cell Cycle Indicator (CA)5 (FUCCI(CA)5), a fluorescent live cell cycle reporter construct. The system introduces two fluorescent, truncated forms of Geminin and Cdt1, which are produced and recycled at specific rates during the cell cycle. Additionally, apoptosis can be directedly monitored during the experiment using a stably expressing FUCCI(CA)5 line of U2-OS cells. During the cell cycle, the reporter operates predictably with each phase because these are produced and degraded at the same rates as the functional versions naturally expressed in the cell. When apoptosis occurs, this cycle is disrupted with the cell cycle rapidly shutting down. Typically, this will manifest visually as the fluorescence staling within the cell cycle, followed by dramatic morphological changes to the nucleus, which is clearly visible due to the localization of the reporters to the nucleus⁶. This allows visual identification of each phase in single cells over time (Figure 2). Reversible electroporation causes changes to intracellular conditions and a 180 V exponential decay pulse was selected for investigation. This pulse was selected to limit cell death as much as possible, while ensuring electroporation in the majority of the population. This was determined based on prior research investigating optimal electroporation pulse parameters for six mammalian cell lines. The energy density of a 180 V pulse was slightly lower than the optimal transfection parameters, providing better cell viability with high poration⁷. The effect of the pulse was studied in unsynchronized cells and S-phase synchronized cells using a thymidine double block with and without PEF application (Figure 3). The results indicated a decrease in average length for S and G2 compared to control. This indicated that the S-phase was sensitive to PEF exposure resulting in a faster cell cycle. This demonstrated that the electroporation cytometry system was able to combine the delivery of PEFs with long-term live cell imaging for studying the cell cycle. While we used this platform to observe cell progression through the cell cycle in response to PEFs, this is a useful tool for studying a variety of applications and conditions for PEFs and cell culture.

Protocol

1. Cell culture preparation

1. Prepare the stable line using reversible electroporation with the following parameters: 100 µL of resuspended U2 OS cells (6 × 10⁶ cells/mL) with 5 µg of plasmid DNA in a 0.4 cm cuvette, and a 180 V square-wave pulse for 10 ms.
2. D-print the custom chamber design using any appropriate 3D printer and biocompatible plastic such as polylactic acid (PLA) (see Supplemental File 1). Disinfect the completed chamber using ethanol (70%), and store in a sterile container prior to use.
3. Culture U2-OS FUCCI(CA)5 cells in a 10 cm plate with 10 mL of Dulbecco's Modified Eagle Media (DMEM)-High Glucose containing 10% Fetal Bovine Serum and 1% PenStrep and incubate at 37 °C and 5% CO₂.
4. When the culture reaches 80% confluency, aspirate the media, wash the cell culture with Phosphate-buffered Saline (PBS), and dissociate the cells using 1 mL of 0.25% Trypsin for 1.5 min.
5. Neutralize the trypsin by resuspending the cell culture in 5 mL of DMEM-High Glucose (containing 10% FBS and 1% PenStrep).
6. From the resuspended cell culture, obtain a 100 µL sample and place it in the electroporation cytometry system chamber on the bottom glass coverslip. Ensure that the chamber is 30% confluent after introducing the sample. If the sample appears to be overconfluent prior to incubation, aspirate the sample and lower the sample volume.
7. Slowly, add 4 mL of DMEM-High Glucose (containing 10% FBS and 1% PenStrep) to the far end of the chamber, preventing the liquid from flushing out the sample.
8. Place a standard microscope slide on top of the chamber and incubate the chamber at 37 °C and 5% CO₂ for 24 h, ensuring the culture has fully adhered to the chamber bottom. Optionally, use Hoechst 33342 (1 µg/mL) to stain cell nuclei for easier downstream cell quantification as well as apoptosis. Perform the stain 30 min prior to time-lapse microscopy.

2. Microscope preparation

1. First, turn on an inverted fluorescent microscope with environmental controls and chamber, allowing for conditions to stabilize prior to performing electroporation.
   NOTE: Any inverted fluorescence microscope with an environmental system that can accept the 25 mm x 55 mm x 16 mm chamber slide. The design was originally designed to work on the referenced inverted microscope (see Table of Materials) and the corresponding environmental system.
   Heating of the stage can cause a drift in the image due to metal expansion.
2. While the environmental system is reaching the optimal temperature, connect a Digital Multimeter to the pulse generator capacitor and high-voltage switch using suitable electric cables. To follow this protocol, use "alligator" clip cables to allow for rapid assembly and disassembly with the electroporation chamber.
3. Next, select 20x magnification and using the microscope's software, ensure that FITC (GFP) and A594 (TexRed/mCherry) channels (optionally, DAPI channel for Hoechst 33342) are selected corresponding to the two fluorescent proteins in the FUCCI(CA)5 system. Use low exposure settings (typically <0.05 s) to avoid photo damage over long-term time-lapses.
4. Optimize the image focus and quality by taking test images prior to beginning the time-lapse.
   NOTE: Fluorescence should be only as bright as necessary for color visualization.
5. Program the time-lapse to capture 3 - 6 viewpoints every 10 min for 24 h to obtain a high volume of cell cycle data per time-lapse while mitigating photo damage.
   NOTE: The length of time may vary depending on the timescale of the cellular activity.
6. Ensure that auto-focus is enabled for the A594 channel for best results tracking the cell culture during the time-lapse.
7. Leave the software in standby mode until electroporation is performed. Once the pulse is delivered, immediately activate the time-lapse to ensure the highest possible data accuracy.

3. Electroporation

1. Place the electroporation cytometry chamber into the microscope with an appropriate environmental chamber. Prior to electroporation, leave the chamber closed with the environmental system running to ensure the cell cycle is not affected.
2. Attach a Digital Multimeter to the pulse generator and connect the pulse generator to the chamber electrodes using alligator clip cables by opening the environmental chamber.
3. Charge the capacitor in the pulse generator to 180 V and quickly discharge the pulse into the chamber until the Digital Multimeter reads 0 Volts.
4. Remove the electrical connections to the chamber and replace the lid as quickly as possible to ensure proper culturing conditions.
5. Immediately activate the time-lapse once the microscope environmental system is in place.

4. Time-lapse data acquisition

1. After the time-lapse has been completed, use the microscope software to save the time-lapse as a single video based on the images captured over the length of the time-lapse. Improve the visual quality at this stage to assist with manual quantification.
   NOTE: Any frame rate can be selected; however, 6 frames/s provides a moderate replay speed with 1 video second resulting in 1 h of real time.
2. Once the time-lapse video is prepared, track the cell phases over time by observing the changes in FITC and A594 signals

Results

Using the FUCCI system (Figure 2), visual cell cycle data were obtained by measuring the length of time for which each color was expressed by the cell as a result of protein expression and unbiquitination. Based on the results from the unsynchronized and synchronized experiments (Figure 3), it was evident that S-phase PEF exposure upregulated the cell cycle. This was evidenced by the decrease in average phase length, indicating a faster cell cycle. Moreover, it ...

Discussion

When performed correctly, cell cycle phase data can be quantified for high volumes of single cells for periods up to 24 h. This protocol yielded comparable cell cycle data to alternative methods such as FACs. This is a commonly used method whereby cells become fluorescently labeled as they progress through the cell cycle. After incubating for at least 24 h, they are sorted by fluorescent intensity, correlating to the amount of the population in a particular phase⁸. Prior research using this method...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chamber Slide (3D Printed/Assesmbled)	N/A	N/A
Culture Plate (10 cm)	Sarstedt	83.3902
Delta Vision	Olympus	N/A	Alternative microscopes models can be used as well. Ensure the stage and environmental system can accommodate the electroporation chamberslide (55 mm x
DMEM – High Glucose	Gibco	11965092
Fetal Bovine Serum	ThermoFisher	A5256701
i3 MK3S 3D Printer	Prusa	N/A	Alternative printer models may be used that can use STL files.
Pen Strep	ThermoFisher	15070063
Phosphate Buffered Saline	Gibco	10010031
tFUCCI(CA)5	Addgene	153521
Trypsin	Sigma-Aldrich	9002-07-07
U2-OS Cell Line	ATCC	HTB-96