Enhancing Cell Viability and Efficiency of Plasmid DNA Electrotransfer Through Reducing Plasma Membrane Permeabilization

Abstract

Background: Pulsed electric field has been widely used to facilitate molecular cargo transfer into cells. However, the cell viability is often decreased when trying to increase the electrotransfer efficiency. We hypothesize that the decrease is due to electropermeabilization of cell membrane that disrupts homeostasis of intracellular microenvironment. Thus, a reduction in the membrane permeabilization may increase the cell viability.

Materials and Methods: Different compounds were supplemented into the pulsing buffer prior to electrotransfer for reduction of cell membrane damage. Extent of the damage was quantified by leakiness of the membrane to a fluorescent dye, calcein, preloaded into cells. At 24 hours post electrotransfer, cell viability and electrotransfer efficiency were quantified with flow cytometry.

Results: The cell viability could be substantially increased by supplementation of either type B gelatin or bovine serum albumin (BSA), without compromising the electrotransfer efficiency. The supplementation also decreased the amount of calcein leaking out of the cells, suggesting that the improvement in cell viability was due to the reduction in electrotransfer-induced membrane damage.

Conclusion: Data from the study demonstrate that type B gelatin and BSA can be used as inexpensive supplements for improving cell viability in electrotransfer.

Introduction

Pulsed electric field can facilitate delivery of molecular cargo into cells. The delivery method, called electrotransfer or electroporation in the literature, has been widely used in different applications ranging from gene delivery, genome editing, and treatment of diseases.^1–7 Compared with other approaches to cargo delivery, electrotransfer is inexpensive, flexible, and safe in the clinic.² However, a drawback of the method is that it often causes cell death.

Mechanisms of the cell death are complicated. It is known that electrotransfer causes damage of the plasma membrane that allows unregulated exchange of molecules between the extracellular medium and cytosol.^8–10 The exchange disrupts cellular homeostasis and allows cellular uptake of toxic chemicals generated by the pulsing.¹¹ In addition, electrotransfer can disrupt the cytoskeleton,^12–14 alter transmembrane potential,¹⁵ and cause cytochrome c release from mitochondria.^16,17 All these changes can trigger cell apoptosis.

One of the approaches to reducing the cell death is to decrease the electrotransfer-induced membrane permeabilization. However, the permeabilization is known to be the dominant mechanism for small-molecule cargos to enter the cells.^18–21 Thus, any decrease in membrane permeability will reduce the delivery of small-molecule cargos.

For macromolecular cargos, such as DNA, RNA, and proteins, the transient pores in the plasma membrane induced by electric pulsing are either too small compared with the size of the cargos or too short for transmembrane transport.^1,2,19 Previous studies have shown that the efficiency of macromolecule electrotransfer depends on endocytosis,^2,12,22–24 suggesting that cellular uptake and cytoplasmic transport of macromolecular cargos are active processes, and that they may not be sensitive to electropermeabilization of the plasma membrane. Therefore, we hypothesize that a reduction in the electropermeabilization can increase the cell viability but not necessarily decrease the electrotransfer efficiency for macromolecular cargos.

To test the hypothesis, we screened nontoxic compounds that could effectively reduce the membrane permeabilization and thus increase the cell viability, and we discovered type B gelatin to be the best candidate under the screening conditions. When supplemented into the pulsing buffer, it could significantly improve both the cell viability and the electrotransfer efficiency.

Materials and Methods

Chemicals

Hepes-buffered saline (HeBS) (20 mM Hepes, 137 mM NaCl, 5 mM KCl, 6 mM dextrose, 0.7 mM Na₂HPO₄, pH 7.0) was used as the electrotransfer buffer unless mentioned specifically in the Results section, and the following chemicals were supplemented into the buffer before electrotransfer: type A gelatin (cat no. 2500) or type B gelatin (cat no. 9391) powder, bovine serum albumin (BSA), hyaluronic acid, fibronectin, dextran sulfate (MW = 200,000 or 500,000), dextran 70 (MW = 70,000), and glutathione (GSH). They were purchased from Sigma-Aldrich. Calcein acetoxymethyl ester (AM) was purchased from Thermo Fisher Scientific. The stock solution of gelatin (2%) was prepared with water and autoclaved at 121°C and 1.05 kg/cm² for 15 min before use. The plasmid DNA (pDNA) encoding enhanced green fluorescent protein (EGFP) (pEGFP-N1; Clontech) was used in all electrotransfer experiments.

Cell culture

Human colorectal carcinoma cells (HCT116; ATCC) were cultured at 37°C with 5% CO₂ in McCoy's 5A medium (Thermo Fisher Scientific) supplemented with 10% bovine calf serum (SeraDigm) and 1% penicillin-streptomycin (Thermo Fisher Scientific). They were passaged every 1–2 days before use.

Electrotransfer procedure

Approximately 5 × 10⁶ cells were plated in a 10-cm culture dish at 24 h before the experiment, allowing them to grow to 75–90% confluency. In the experiment, cells were detached by treatment with 0.25% trypsin-ethylenediaminetetraacetic acid (Invitrogen), mixed with 10 mL complete culture medium for trypsin neutralization, and harvested by centrifugation. Then, the cell pellets were washed with Dulbecco's Phosphate-Buffered Saline (DPBS), resuspended in 1 mL HeBS buffer (with or without supplements) at a density of 10⁷ cells/mL, and mixed with 30 μL pDNA solution (1 μg/μL). One hundred microliters of the mixture with 1 × 10⁶ cells and 3 μg of pDNA were loaded into a disposable 4-mm gap aluminum cuvette (Bio-Rad).

The cells were treated with an electric pulse sequence generated by the BTX-ECM830 Square Wave Electroporation System (Harvard Apparatus). Each sequence consisted of six pulses at 200 V/4 mm, 5 ms duration, and 1 Hz frequency. After pulsing, the samples were incubated at room temperature for 10 min, seeded in 6-well plates containing 2 mL of fresh culture medium, and cultured at 37°C with 5% CO₂.

Electrotransfer efficiency and cell viability

They were evaluated with flow cytometry at 24 h after pulsing. The details have been previously described.^{2,11,12,22,25} In brief, cells in each well were collected, resuspended in 200–300 μL medium, and stained with propidium iodide (PI) at a concentration of 5 mg/mL. The samples were loaded into a flow cytometer (BD FACSCanto II, Becton Dickinson) equipped with a 488-nm laser for simultaneous excitation of EGFP and PI. Single-cell populations were separated by using front and side light scattering as independent variables. Compensation was set to resolve emission spectra overlap between the two detection channels.

To determine the electrotransfer efficiency and the cell viability, the single-cell populations were collected for 30 s at the fast mode. The data were used to calculate three parameters: electrotransfer effectiveness (eTE), transgene expression level, and cell viability. The eTE was defined as the percentage of PI-/EGFP+ population among the total viable (PI-) cells; and the expression level was quantified as the geometric mean (GM) of the green fluorescence intensity. The cell viability was defined as the number of live cells (i.e., PI-) in an experimental group normalized by that in the matched control group. FlowJo software was used in all data analysis.

Plasma membrane permeabilization assay

To determine the extent of cell membrane permeabilization, the cells were preloaded with a fluorescent marker of cytosol, calcein-AM. The permeabilization would cause calcein to leak out of the cells, thereby decreasing the fluorescence intensity. In the experiment, 1 mM stock solution of calcein-AM was prepared with DPBS, and it was mixed with 1 mL suspension with 3 × 10⁶ cells at a concentration of 5 μM. After incubation at the room temperature in a dark space for 30 min, the cells were washed twice with 2 mL fresh DPBS, and they were used immediately in electrotransfer experiments. Within 15 min after pulsing, the samples were loaded into the flow cytometer, and single-cell populations were collected for 30 s at the fast mode to determine the histogram of the fluorescence intensity for each sample.

Statistical analysis

Error bars in all figures represent the standard error of the mean (SEM). Differences between two different groups were evaluated with the Mann–Whitney U test (Prism, GraphPad Software). They were considered statistically significant if p < 0.05.

Results and Discussion

Effect of chemical supplements on cell viability and electrotransfer efficiency

The viability of cells was decreased after the electrotransfer of pDNA (Fig. 1). The observation was consistent with those in other studies.^12,22,25 To increase the viability, we supplemented different compounds into the pulsing buffer (i.e., HeBS) before the application of electric pulses (see the discussion below), and we identified type B gelatin and BSA to be the most effective compounds for prevention of cell death.

FIG. 1.

Effects of gelatin and BSA supplementation on cell viability. The compounds were supplemented into the pulsing buffer at the concentrations of 0.2% for both type A and type B gelatin, and 1% for BSA. Two control groups (Ctrl) are included for comparisons, in which the cells were either electrically NP or pulsed in the buffer without any of the compounds (untreated). The data were normalized by the NP Ctrl group. *p < 0.05, n ≥ 5 (number of independent repeats of the experiments), Mann–Whitney U test. BSA, bovine serum albumin; NP, non-pulsed.

At their optimal concentrations, type B gelatin and BSA increased the cell viability by 3.8- and 5.2-fold, respectively, compared with that in the untreated control group (Fig. 1), but the difference in cell viability between the BSA and the type B gelatin groups was statistically insignificant (p > 0.05). Supplementation of the pulsing buffer with type A gelatin at the same concentration had only a minor effect on the viability, compared with the untreated control group (p > 0.05) (Fig. 1). The lack of effects on the viability could not be improved by increasing the concentration of type A gelatin from 0.2% to 4% (data not shown).

In addition to the viability, we quantified the eTE and the expression level of the transgene (i.e., EGFP). We observed that both type B gelatin and BSA supplementations could increase the eTE (Fig. 2) but had insignificant effects on the expression level (Fig. 3). The BSA data were consistent with those obtained in a previous study.²⁶ Taken together, our data demonstrated that type B gelatin and BSA could be used to improve the cell viability without compromising the electrotransfer efficiency although mechanisms of the improvement were still elusive.

FIG. 2.

Effects of gelatin and BSA supplementation on eTE. The experimental details are the same as those in Figure 1, except that the eTE data from the same experiments are reported here. No cells were observed to express EGFP in the non-pulsed control group (data not shown). *p < 0.05, n ≥ 5, Mann–Whitney U test. EGFP, enhanced green fluorescent protein; eTE, electrotransfer effectiveness.

FIG. 3.

Effects of gelatin and BSA supplementation on EGFP expression level. The expression level was quantified as the GM of the fluorescence intensity (arbitrary unit) by using flow cytometry. The experimental details are the same as those in Figure 1, except that the intensity data from the same experiments are reported here (n ≥ 5). No cells were observed to express EGFP in the non-pulsed control group (data not shown). GM, geometric mean.

Potential mechanisms of improvement in cell viability

To understand the mechanisms, we first investigated whether gelatin could block the permeabilization of the plasma membrane induced by electrotransfer. The blockage would reduce exchange of chemicals across the cell membrane, thereby inhibiting the disturbance of the intracellular environment. In the experiment, we preloaded the cells with calcein-AM at ∼30 min before the electrotransfer, and we measured the histogram of fluorescence intensity in six different cell populations within 15 min post-electrotransfer.

The data indicated that the background fluorescence in cells was low (Fig. 4A). Cellular uptake of calcein-AM increased the fluorescence intensity in the majority of cells (Fig. 4B). Treatment of cells with electric pulses alone could permeabilize the plasma membrane, as indicated by the leftward shift of the intensity histogram and the decrease in the mean intensity (Fig. 4C), due to the reduction in the amount of calcein leaking out of the cells. When pDNA was electrotransferred into the cells with the same electric pulse sequence as that in the pulsing alone group, the fluorescence intensity was further decreased (Fig. 4D). These data indicated that electrotransfer caused more membrane damage than electric pulsing alone; and the additional damage was mediated by the direct interactions between pDNA and the membrane.^18,24

FIG. 4.

Effects of type B gelatin and BSA supplementation on electropermeabilization of plasma membrane. Some cells were loaded a fluorescent dye, calcein, before application of electric pulses. The membrane permeabilization induced by the pulses or electrotransfer of pDNA was measured by changes in the fluorescence intensity due to leaking out of calcein from the cells. The panels show (i) the GM of fluorescence intensity, (ii) the histogram of the fluorescence intensity of cells, and (iii) the percent of cells in low- and high-intensity regions of the histogram. (A) The cells were not exposed to any treatments. (B) The cells were pretreated with calcein-AM but not used in electrotransfer experiments. The histogram was used to define the low- and high-intensity regions indicated by the horizontal bars. (C) The cells were pretreated with calcein-AM, and then they were electrically pulsed in the buffer without pDNA. (D) The cells were pretreated with calcein-AM, and then they were electrically pulsed in the buffer with pDNA. (E) The details are the same as those in (D), except that the pulsing buffer was supplemented with 0.2% type B gelatin. (F) The details are the same as those in (D), except that the pulsing buffer was supplemented with 1% BSA. AM, acetoxymethyl ester; pDNA, plasmid DNA.

When type B gelatin or BSA was supplemented into the pulsing buffer, the electrotransfer-induced decrease in fluorescence intensity was blocked (Fig. 4E, F), indicating that these compounds could inhibit calcein leaking out of the cells. The same conclusion could be made with the GM of fluorescence intensity (arbitrary unit). In the six experimental groups shown in Figure 4A through F, the mean ± SD of the GM from three independent repeats of the experiment were 8.5 ± 0.1, 20,100 ± 1217, 5421 ± 738, 3735 ± 54, 9888 ± 495, and 6585 ± 1111, respectively. Plasma membrane damage may cause cell aggregation. Our study showed that the aggregation could also be prevented when type A or type B gelatin was supplemented into the pulsing buffer (Fig. 5).

FIG. 5.

Effects of gelatin supplementation on cell aggregation. The images were acquired under a light microscope equipped with a 10 × objective at 30 min post-electrotransfer. (A) Cells were not exposed to any treatments. (B) Cell aggregation was observed after electrotransfer of pDNA. (C) The aggregation was significantly reduced in the group where the pulsing buffer was supplemented with type B gelatin at 0.2% concentration. (D) The type A gelatin at 0.2% concentration had the same effects as type B gelatin on the reduction of cell aggregation.

Next, we investigated whether type B gelatin could improve the cell viability by facilitating cell adhesion to the culture plate post-electrotransfer. In the experiment, we compared two situations. The first one is discussed earlier (Fig. 1), where type B gelatin was supplemented into the pulsing buffer, and the cell suspension in the buffer was transferred to fresh medium for cell culture post-electrotransfer. In the second situation, type B gelatin was supplemented into the fresh medium rather than the pulsing buffer. In both situations, type B gelatin was present in the culture medium at the same concentration. Our data showed that adding the gelatin into the culture medium had little effects on the cell viability (Fig. 6), suggesting that gelatin-mediated improvement in cell adhesion had little effects on cell viability. To further confirm the data, we supplemented the pulsing buffer with fibronectin at various concentrations (0.01–0.1%). To our surprise, the supplementation reduced the cell viability (data not shown).

FIG. 6.

Effects of timing of gelatin supplementation on cell viability. The pDNA was electrotransferred into cells suspended in 100 μL buffer containing no gelatin (Untreated Ctrl) or 0.2% type B gelatin (Treated in EP Buffer). Then, the suspensions were transferred to the medium, and the cells were cultured for 24 h. The same data have also been shown in Figure 1. In a separate experiment, the electrotransfer was performed in 100 μL buffer without gelatin supplementation. Then, the suspensions were transferred to the medium supplemented with type B gelatin (Treated Post EP). The final gelatin concentration in the medium was the same as that in the second group. After the cells were cultured for 24 h, the viability was measured with flow cytometry. *p < 0.05, n ≥ 4, Mann–Whitney U test.

Type A and type B gelatins have different isoelectric points, and they are positively and negatively charged in HeBS buffer, respectively.²⁷ Therefore, we wondered whether the negative charge of type B gelatin might play a role in improving the cell viability. To investigate this possibility, we supplemented other negatively charged macromolecules into HeBS buffer for electrotransfer. They include hyaluronic acid at concentrations of 0.05% to 0.2% and dextran sulfate with a molecular weight of 200,000 or 500,000 at concentrations of 0.001% to 0.5%. At 24 h post-electrotransfer, we observed that neither hyaluronic acid nor dextran sulfate could improve the cell viability (data not shown), suggesting that the electric charge was unlikely to play a key role in the type B gelatin-mediated improvement in cell viability shown in Figure 1.

Gelatin and its hydrolysates may have antioxidant properties;²⁸ it is well known that electric pulsing can induce membrane lipid oxidation.^29,30 Therefore, we investigated whether other antioxidants, such as GSH, could be used to increase the cell viability. In the experiment, GSH was supplemented into the pulsing buffer at different concentrations (0.00001–0.01%) for electrotransfer, and the cell viability was measured at 24 h post-electrotransfer. We found that the supplementation did not improve the viability at all GSH concentrations tested (data not shown), suggesting that the improvement in cell viability shown in Figure 1 was not due to the antioxidant property of gelatin.

There are two limitations in the study. First, we only presented the effects of gelatin and BSA on cell viability by using the data obtained with one cell line and one experimental protocol, although the BSA data are consistent with those previously observed.²⁶ It is expected that the supplementation of the pulsing buffer with gelatin or BSA should have insignificant effects on cell viability if in the control group, its value is close to 100%, or the plasma membrane is severely damaged. Second, we do not know at present how gelatin or BSA improved cell viability although we have excluded a few potential mechanisms discussed earlier. These limitations need to be addressed in future studies.

Conclusion

This study demonstrated that type B gelatin and BSA could improve cell viability and electrotransfer efficiency when they were supplemented into the pulsing buffer. The improvement was due to the inhibition of electrotransfer-induced plasma membrane damage. Although the exact mechanisms of the inhibition are still elusive, type B gelatin and BSA may be used practically as inexpensive supplements for improving electrotransfer.

Authors' Contributions

Conceptualization: Y.W. and F.Y.; methodology: Y.W., C.C., and F.Y.; investigation: Y.W., C.C., L.W., and F.Y.; data curation: Y.W., C.C., and L.W.; writing and editing: Y.W., C.C., and F.Y.; visualization: C.C. and F.Y.; and project administration: F.Y.

Disclaimer

This article has been submitted solely to this journal and is not published, in press, or submitted elsewhere.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

The work was supported partly by a grant from the National Institutes of Health (GM130830).