ID: 2249

• Title:
  The impact of cell density on the electrical parameters of pulsed electric field applications
  
  Authors:
  

  Caldeira, Viviana1,2; Calado, Cecília1; Redondo, Luís1,2
  
  1. R&D Laboratory in Health & Engineering, Lisbon School of Engineering/ISEL, Lisbon, Portugal. 
  2. Pulsed Power Advanced Applications Group, Lisbon School of Engineering, GIAAPP/ISEL, Lisbon, Portugal.
  Correspondence:  lmredondo@deea.isel.ipl.pt
  

  
  
  Abstract:
  Pulsed Power (PP) is one of the most effective and powerful ways of delivering energy, it is used in particle accelerators, in radar systems, as well as its application in industrial processes. Pulsed Electric Fields (PEF) involves exposing materials to energy given by a PP generator. The use of PEF in biological systems – Bioelectrics – it is quite complex. PEF induce electroporation, increasing membrane permeability and allowing ions and water molecules passage and its typically applied in a cuvette-based batch system. Monitoring electrical parameters of PEF, such as electric field strength, pulses number and specific energy, are crucial. Nevertheless, there is a common gap in PEF literature, there's no reference of cell density impact. Thus, this experimental work aims to evaluate the impact of cell density and its interference on the electric fields between the electrodes studying the results on cell viability and metabolomics, hypothesizing that cell density can be crucial for in vitro PEF experiments. In this experiment were used immortalized lines of L929 fibroblast cells, maintained at 4ºC to minimize metabolic activity, and sterilized electroporation cuvettes with DPBS as conductive solution due to conductivity (5.5 mS/cm) and non interfering ions. The experiment consisted of five groups, ranging from 1x106 to 5x104 cells per 800µL of DPBS, including control groups. The electrical parameters chosen were, ton=5us, f=1Hz, U=4kV, P=5, and E=10kV/cm. Statistical analysis (AV, SD) and Analysis of Variance (ANOVA), were performed for cell density and viability evaluations (by trypan blue assay). Fourier-transform infrared spectroscopy (FTIR) accessed metabolomics. samples were analyzed immediately after PEF experiments (T=0) and after 72 hours in culture (T=72). Subsequent data processing was performed with OPUS, Unscrambler X, and Excel software, for Principal Component Analysis (PCA). Parameters such as specific energy (Ws), temperature increase (∆T), and current (A) were also monitored after each experiment. It was found that higher cell densities, particularly between 1x106 and 5x105 cells per 800µL, resulted in higher cell viability. The analysis of electrical current and temperature showed variations across different cell groups, with group D and E experiencing the most significant increases in current and temperature (296A and 12.8ºC at P=5). Viability analysis immediately after PEF exposition revealed that group A had the highest average viability (79%) and the lowest percentage of lysed cells (below 8%), when compared to Group D and E exhibited the lowest viability (23% and 18% respectively) and high levels of cell lysis. After 72 hours in culture, cells exposed to PEF demonstrated a viability above 71%, being the most promising groups, A and B. ANOVA analysis showed significant differences between conditions with and without PEF. Culture media after 72 hours showed non-significant differences in group A and B, indicating recovery comparable to the control groups. PCA of normalized second derivative spectra supported the impact of cell density on cell metabolism, demonstrating clustering of samples based on cells density at T=0. Additionally, the PCA analysis of culture media after 72 hours indicated distinct clusters based on the number of cells. The statistical analysis, p-values (less than 10%), and PCA demonstrated consistent results, confirming assay reproducibility, and supporting the initial hypothesis regarding the impact of cell density on PEF applications. The findings revealed that, among the evaluated conditions higher cell densities exhibited lower levels of cell lysis, higher cell viability, and better cell proliferation even after 72 hours of PEF application, with minimal temperature increase during PEF. These results provide valuable insights for future research, suggesting that further studies are necessary to optimize PEF applications. This study serves as a foundational step towards understanding PEF relations and cellular responses, paving the way for future advancements in this field.
  
  Keywords:
  
  
  Refs:
  
  
  Topic 1:
  12. Biomedical applications
  
  Topic 2:
  1. Biological responses (molecular, subcellular, cellular and intercellular)