Novel electroporation microchip with field constriction enhances transfection efficiency and survival rates of feline embryos

Microchip device structure and experiment setup

The complete assembly of the microchip device is illustrated in Fig. 1A. The electroporation microchip device features a dual-layer PDMS chamber design (top and bottom). All components are transparent, allowing for easy visual observation, and they are not permanently bonded but rather fixed together by bolt compression at the four edges. This design permits disassembly for cleaning and reassembly for repeated use, as shown in Fig. 1B. Microfluidic inlet and outlet ports are attached to the device and connected to a syringe pump (NE-1000; New Era Pump Systems, Farmingdale, NY, USA) to control the movement of embryos within the microchip. The process involves trapping and stabilising the embryo at the orifice, followed by releasing it by reversing the fluid flow direction, facilitating the replacement of the next embryo. The flow rate is critical to avoid damaging the embryo; in this study, a rate of 10 to 15 µL/min was used for trapping, with a slower rate (1–5 µL/min) applied during electroporation for stabilisation. The inlet and outlet ports also function as the device’s electrodes, connected to a function generator (AFG3021B; Tektronix, Beaverton, OR, USA) and an impedance analyser (E4990A; Keysight, Santa Rosa, CA, USA). This significantly wider device’s electrode separation (4 mm compared to 0.26 mm in conventional devices) resulted in a lower current density and reduced Joule heating. Additionally, the small orifice in the microchip increased electrical impedance, which further minimises temperature effects and could enhance cell viability during electroporation. Unlike conventional devices (Fig. 1C), a polyester film with a small orifice is positioned between the chambers, serving as an insulating barrier that creates field constriction, as shown in Fig. 1D. This configuration ensures that the majority of the electrical field lines are focused on the cell membrane at the orifice⁷. This approach reduces leakage current passing through the gap between the embryo and the orifice edge, helping to minimise variations in the electroporation results. It is worth noting that a slight gap between the embryo and the orifice should not impact electroporation, as previously demonstrated³¹. Without field constriction, membrane breakdown typically occurs over a larger portion of the cell membrane³². Therefore, this microchip aids in the localised creation of transient pores on the embryo membrane at the orifice, facilitating targeted particle delivery. The experimental setup is illustrated in Fig. 1E. Embryos are carefully introduced into the upper chamber fluid and trapped at the orifice using a water flow system. The syringe pump and function generator are connected to the electrodes and controlled by the host, while data from the impedance analyser are monitored via computer. The experiments were conducted under a microscope (IX73; Olympus, Tokyo, Japan) equipped with a fluorescence system for observing dynamic changes in the embryos.

Electroporation rate analysis

Figure 2A shows that the orifice on the thin insulator plate of the microchip has a diameter of approximately 100 μm; this is smaller than the size of cat embryos, which range from 150 to 170 μm, including their zona pellucida. This size allows for proper contact between the embryo and the orifice, enabling the creation of an intense electrical field at the cell membrane and leading to effective electroporation. To evaluate the efficacy of electroporation using this microchip, pulses of varying amplitudes were applied. The Yo-Pro-1 fluorochrome permeates the embryo only when the membrane is porated. Thus, fluorescence intensity serves as a direct indicator of electroporation efficiency, with higher intensity reflecting greater membrane poration. The transient pores formed on the embryo membrane allowed the fluorescent dye to diffuse into the embryo, resulting in green fluorescence. Images of embryos under the fluorescent microscope were captured before (Fig. 2B) and after (Fig. 2C) pulsing while the embryo was positioned at the orifice. In the negative control group (no pulsing), no fluorescence was observed, confirming that green fluorescence in the experimental groups resulted from successful membrane poration. The green fluorescence gradually intensified and reached saturation within 3 min after pulsing, with Yo-Pro-1 dye dispersing throughout the cytosol of the electroporated embryo. The presence of Yo-Pro-1 dye indicates electroporation efficiency through membrane permeability during electrical pulses. However, fluorescent intensity does not correlate with long-term embryo viability, as high voltage can compromise health despite increased fluorescence. Our findings suggest that impedance analysis, which assesses membrane resealing ability, is a better predictor of embryo development. Successful electroporation depends on optimal electrical stimulation and rapid pore resealing to stabilise the cell environment, thereby ensuring long-term embryo viability and development³³. Moreover, there were no apparent morphological alterations in the embryos after receiving pulses through the orifice across all voltage groups. Figure 2D compares the fluorescence intensity of embryos between the novel microchip and conventional platforms. The fluorescence intensity of Yo-Pro-1 increased with rising voltage, indicating enhanced membrane perforation efficiency. Under low-voltage conditions (10–15 V), the fluorescence intensity of embryos using the microchip was significantly higher than that of the conventional platforms (p < 0.05). However, the fluorescence intensity at 20 V was not significantly different between the novel and conventional groups (p = 0.097). This suggests that the microchip improves transfection efficiency, particularly under low-voltage conditions. Based on this evidence, 15 V was selected as the standard condition for subsequent experiments. The fluorescence intensity showed a remarkable increase in the novel microchip groups, with values ranging from 40 to 460% compared with the conventional groups. The results demonstrated that the microchip with 15 V achieved a 69.5% higher electroporation rate compared to conventional devices (p < 0.05).

Electroporation rate analysis and comparison of Yo-Pro-1 fluorescence intensity. (A) Bright field image of the orifice without a cat embryo. (B) Fluorescent microscope image of the embryo positioned on the orifice before electroporation. (C) The embryo on the orifice showing green fluorescence (Yo-Pro 1) at 3 min post-electroporation. Arrowheads in (B) and (C) indicate the embryo, including its zona pellucida. (D) Comparison of Yo-Pro-1 fluorescence intensity at different voltages (10, 15, and 20 V) between conventional and novel platform groups (*p < 0.05).

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Assessment of embryo viability

To verify the viability of embryos post-electroporation, EthD-1 was used to evaluate embryo viability. This fluorescent dye is a cell-impermeant viability indicator that enters embryos with damaged membranes. In viable embryos, EthD-1 is excluded, resulting in no fluorescence as shown in Fig. 3A. However, in embryos with damaged membranes, the dye enters and binds to DNA, emitting bright red fluorescence as visualised in Fig. 3B. The percentages of viable embryos in the microchip and conventional groups (n = 50 embryos/group) under different voltages (0 as control, 10, 15, and 20 V) at 2 h following electroporation are illustrated in Fig. 3C. A decrease in viability was observed in both the novel microchip and conventional groups as the pulse amplitude increased, suggesting that the electroporation pulse adversely affects the embryo membrane. At higher voltages, larger and more persistent pores form in the membrane, making resealing difficult. This can lead to increased calcium influx, osmotic imbalance, and activation of stress pathways, potentially leading to cell death³⁴. Compared with the control group (0 V), a significant decline in viability was observed in the conventional group at 15 V and 20 V (92% and 84%, respectively), whereas embryos in the microchip group exhibited a decrease only at 20 V (90% viability). Additionally, when comparing the microchip and conventional groups at the same pulse amplitude, the microchip provided a significantly higher viability rate (100% vs. 92%, p < 0.05) at 15 V. However, the 10 V and 20 V condition groups did not show significant differences. These findings suggest that the microchip’s field constriction design minimises membrane damage by focusing the electric field precisely at the membrane contact point, thereby limiting the affected area. This approach enables efficient poration at lower voltages, reducing cellular stress and preserving membrane integrity, which could support embryo viability at voltages optimal for transfection^33,35. These findings indicate that applying field constriction to localise the electrical pulse on the cell membrane effectively reduces membrane damage in embryos. Based on these results, 15 V was proposed as the optimal amplitude for further experiments.

Embryo viability and developmental rate following electroporation. (A) Viable cat embryos stained with ethidium homodimer dye 2 h post-electroporation. (B) Non-viable embryos with damaged membranes. (C) Comparison of embryo viability between the novel microchip and conventional groups at different voltages (0 as control, 10, 15, and 20 V) 2 h after electroporation. Developmental rates of embryos post-electroporation, including (D) cleavage rate on day 2 and (E) blastocyst rate on day 7 after IVF, calculated relative to the total number of experimental embryos. Significant differences are indicated (*p < 0.05, **p < 0.001).

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Assessment of embryo developmental rate

The results regarding the developmental rate of embryos under different conditions are summarised in Supplementary data 1. Following electroporation, the developmental rate was evaluated on days 2 and 7 post-IVF in a minimum of four replicates. The rate was categorised as the cleavage and blastocyst rate, respectively. Figure 3D shows that the cleavage rate results were not significantly different across all groups. However, the blastocyst rate decreased with increasing voltage in both the microchip and conventional groups, as shown in Fig. 3E. Compared with the control group (cultured without electroporation), no significant differences were observed in the lowest voltage groups (10 V) in both the novel microchip and conventional platforms. A significant reduction in the blastocyst rate was observed in the higher voltage groups, decreasing from 55.41% in the control group to 32.22% and 29.32% (p < 0.05) in the groups using microchip platforms with voltages of 15 V and 20 V, respectively. Conversely, the conventional platform resulted in a marked drop in the blastocyst rate from 55.41 to 25.66% and 23.54% (p < 0.001). This finding suggests a negative correlation between increasing electrical strength and embryo development, consistent with previous reports involving ewe, swine and bovine embryos^36,37,38. Previous studies suggest that electric field strengths impact cellular functions by inducing apoptosis through membrane destabilization. This process also damages critical structures like the cytoskeleton and nuclear membrane, limiting cell survival and replication^39,40,41,42. Although the differences within the same amplitude groups were not statistically significant, embryos from the microchip groups developed into blastocysts at higher rates than those from conventional groups under all voltage conditions. This suggests that employing the field constriction strategy could enable our microchip to improve embryo development.

Impedance measurement after electroporation

To understand the embryo membrane resealing process, real-time impedance measurements were performed over 5 min on embryos following each series of pulses. The initial impedance measurement was taken 15 s after pulse application. Mean resealing times ± standard deviations were analysed from 20 embryos. The embryo membrane resealing time was determined by monitoring changes in relative impedance. The relative impedances (Δ|Z|) denote the impedance signal response after pulses relative to the resting impedance (without embryo). Δ|Z| was normalised by setting the impedance before pulsing as 1 and the impedance without an embryo as 0. The normalised results are plotted in Fig. 4A. After electroporation, a rapid decline in embryo impedance was immediately observed, indicating that electroporation enhances embryo membrane permeability and induces pore formation, leading to decreased impedance⁴³. Subsequently, the impedance gradually recovered and stabilised, although it remained lower than the pre-pulsing impedance. This period of impedance change was defined as the membrane resealing phase, with a measured resealing time of approximately 1.86 ± 0.758 min (range, 0.87–3.25 min). This also corroborates our previous observation that dye saturation occurred within 3 min after electroporation, as observed under a fluorescent microscope. Notably, based on the observed impedance signals, we speculate that the sustained impedance drop post-resealing could be influenced by the disconnect and reconnect process of the cables during electroporation. However, the consistently high viability and developmental rates observed at the same pulse level suggest that the embryo membranes retained functional integrity despite these fluctuations. This suggests that transient interferences may be related to the technical handling of the system.

Impedance measurements and plasmid transfection of cat embryos following electroporation. (A) Impedance measurements of cat embryos at 200 kHz after electroporation at 15 V show that the embryo membrane resealed within approximately 2 min, as represented by the relative impedance value in real-time. (B) Blastocyst embryo expressing GFP reporter from plasmid transfection. (C) Transfected blastocyst embryo with nuclear staining using Hoechst 33342. (D) Merged image showing GFP expression and Hoechst 33342-stained nuclei in the embryo.

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Plasmid transfection

To test the potential use of our microchip platform for transfection, GFP plasmid transfection experiments were conducted on cat embryos. Three experimental groups were evaluated: the negative control group (without plasmid) and the treatment group (with plasmid). using both microchip and conventional devices. Electroporation parameters were selected based on the previously optimised conditions at 15 V. Figure 4B-D shows GFP plasmid expression in embryos at the blastocyst stage. Green fluorescence was observed in the transfected group but not in the control group. The transfection rate of the transfected embryos reached 78.6%, significantly higher than the 53.6% observed in the conventional group (p < 0.05), as evaluated 3 days after transfection. Similar embryo morphology was observed in all groups. The expression of GFP confirmed that our platform successfully delivered macromolecular plasmids (> 10 kb). These findings approved that the microchip is suitable for gene editing with greater effectiveness. However, the blastocyst rate decreased relative to the non-plasmid group, indicating that plasmid DNA can have negative effects on cells. This result is consistent with previous findings that plasmids are toxic to cells when exposed to electroporation pulses. The toxicity is influenced by plasmid size and concentration, high plasmid loads can cause membrane stress and may activate apoptotic pathways, reducing cell viability^44,45.

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• Source: https://www.nature.com/articles/s41598-024-80494-z