ID: 2261

• Title:
  Evaluation of biological responses of 3D multicellular spheroids made of dermal fibroblasts upon RF exposure: assessment of ultrastructural effects of intracellular organelles
  
  Authors:
  

  Kolosnjaj-Tabi, Jelena - 1
  Alberola, Geraldine - 1
  Augé, Sylvain - 2
  Golzio, Muriel - 1
  Dubuc, David - 2
  Grenier, Katia - 2
  Rols, Marie-Pierre - 1
  1)	Institute of pharmacology and structural biology, 205 Route de Narbonne, Toulouse, France
  2)	LAAS-CNRS, 7 Av. du Colonel Roche, Toulouse, France

  
  
  Abstract:
  Background:
  
  To date, the scientific community did not reach a clear consensus if and at which thresholds RF-exposure induces (any) considerable biological effects, so additional studies remain a necessity. While we have previously determined that RF exposure of three-dimensional multicellular spheroids made with HCT-116 (human colorectal cancer cells) does not induce cell membrane permeabilization and does not induce ultrastructural modifications in exposed cells [1], the effect on normal, non-cancerous cells was not studied. In these regards, the objective of this study was to determine if RF exposure causes cell membranes’ permeabilization and ultra-structural alterations of intracellular structures (such as the nuclei, mitochondria, endoplasmic reticulum, etc.) in multicellular spheroids made of normal dermal fibroblasts
  
  
  
  Methods:
  
  A previously described calibrated RF micro-device [1] was used, operating in near field configuration. Three-dimensional multicellular spheroids were made according to the ultra-low attachment procedure, using fibroblasts, derived from healthy human donors’ skin (Icelltis, Toulouse). The spheroids were exposed to different RF parameters (such as frequency, power, or modulation). Exposure was evaluated upon continuous waves (CW) or cycle ratios (CR) of 0.3 or 15% at different SAR values (from 0.43 kW/kg to 7.5 kW/kg) at 2.45 GHz. The spheroids were divided in different groups as follows: 1) Positive control included spheroids treated with pulsed electric field (eight pulses lasting 100 µs, which were delivered with stainless steel parallel plate electrodes, at a frequency of 1 Hz and field intensity of 1.3 kV/cm), and thus induced membrane permeabilization; 2) Negative control included untreated spheroids, not placed in the device; 3) Sham group included spheroids that were placed in the device but were not illuminated and 4) RF-exposed group: spheroids that were illuminated within the micro-device. Cell membrane permeabilization was assessed using propidium iodide. This small molecular probe penetrates the cells when the cellular membrane integrity is disrupted. Upon penetration into the cell, the probe increases its fluorescence yield, allowing to determine if the membrane was altered upon RF exposure. In addition to the evaluation of acute/immediate membrane effects following RF exposure, the general morphology of spheroids was assessed by transmission electron microscopy immediately after exposure and 3 days after exposure (D3). This morphological evaluation allows to detect any potential changes in exposed cells, but also in their immediate surroundings (extracellular spaces) between the cells constituting the multicellular spheroids.
  
  
  
  Results:
  
  Under our experimental conditions the RF exposure did not alter the integrity of cell membranes in normal dermal fibroblasts constituting multicellular spheroids. This is in line with our previous findings observed upon cancerous cells spheroids exposure to RFs [1].
  
  On the level of cellular morphology, immediately after RF exposure, we did no se any overt differences among the different groups of spheroids. Nevertheless, at D3 following the exposure, we evidenced a more abundant extracellular space, filled with cellular debris, microvesicles-like, fiber-like and extracellular smooth endoplasmic-reticulum-like structures.
  
  
  
  Conclusions:
  
  While the fibroblasts’ membrane was not permeabilized upon RF exposure, the morphological alterations in the spheroid indicate that some local biological effects have occurred. Additional functional tests will be performed to assess if these morphological changes alter cellular functions.
  
  
  
  Acknowledgement:
  
  This research was supported by the ANR-15-ASTR-0017 RF METRO CELL grant.
  
  
  
  Keywords:
  3D multicellular spheroids, fibroblasts, RF-exposure, ultrastructure
  
  Refs:
  [1] Kolosnjaj-Tabi, J., Alberola, G., Augé, S., Tamra, A., Dubuc, D., Grenier, K., & Rols, M. P. (2020, December). Evaluation of Cell Membrane Effects After 3D Multicellular Spheroids RF Exposure. In 2020 IEEE MTT-S International Microwave Biomedical Conference (IMBioC) (pp. 1-3). IEEE.
  
  Topic 1:
  1. Biological responses (molecular, subcellular, cellular and intercellular)
  
  Topic 2:
  4. Diagnostics, analytics, experimental techniques