Presentation Information
[P01-100]Measurement of Molecular Diffusion in Three-Dimensional Cellular Tissue Using a Microelectrode
○Gaku Kurogi1, Keita Ebine2, Shotaro Yamada1, Kosuke Ino1, Hitoshi Shiku1, Hiroya Abe1,3 (1. Graduate School of Engineering, Tohoku University (Japan), 2. Graduate School of Life Sciences, Tohoku University (Japan), 3. Frontier Research Institute for Interdisciplinary Sciences, Tohoku University (Japan))
Keywords:
Microelectrode probe,Electrochemical sensor,3-dimensional culture,Spheroids
[Purpose]Understanding the diffusion behavior of substances within biological tissues is important. In drug delivery systems and anticancer therapies, how far drugs reach within tissues strongly affects therapeutic efficacy. However, studies that directly evaluate drug diffusion within biological tissues with spatial and temporal resolution remain limited. In this study, we evaluated the diffusion behavior of substances within three-dimensional cellular structures using spheroids as a biological tissue model combined with electrochemical measurements using a microprobe electrode.
[Method]A platinum microprobe electrode with a diameter of 20 µm was fabricated. MCF-7 spheroids were prepared by culturing the human breast cancer-derived cell line in a 96-well plate for three days and then transferred to a dish for fixation. As model agents, 1 mM ferrocenemethanol (FcCH2OH) and 1 mM potassium ferrocyanide ([Fe(CN)6]4-) were used as hydrophobic and hydrophilic substances, respectively. The microelectrode was inserted into the spheroid from the surface toward the center at 10 µm intervals, and cyclic voltammetry was performed at each position. The electrode was then fixed at the center (approximately 150 µm depth), and chronoamperometry was conducted after the addition of each agent to a final concentration of 1 mM.
[Results]The current decreased as the electrode approached the center of the spheroid, indicating limited diffusion into the inner region. The current of the hydrophilic agent decreased to 9.6% at the center, whereas that of the hydrophobic agent remained at 36.4%, demonstrating a clear difference in permeability. Differences were also observed in the time required for the current to reach a steady state after the addition of each agent. The hydrophobic agent exhibited a slower rise and required a longer time to stabilize.
[Consideration]The hydrophilic and negatively charged Fe(CN)6]4- cannot permeate the cell membrane and mainly diffuses through intercellular spaces, which limits its transport to the spheroid center. In contrast, hydrophobic FcCH2OH can permeate the cell membrane and diffuse via both intracellular and extracellular pathways, resulting in higher accessibility to the inner region. Differences in the rise time and stabilization of the current are attributed to hydrophobic interactions between FcCH2OH and cellular components such as lipid membranes, which transiently retain the molecules within the tissue and delay transport.
[Conclusion]In this study, the diffusion behavior of molecules within three-dimensional cellular structures was evaluated in spatial and temporal aspects. This method is expected to be applicable to the evaluation of the penetration behavior of drugs and chemical components in daily-use products.
[Method]A platinum microprobe electrode with a diameter of 20 µm was fabricated. MCF-7 spheroids were prepared by culturing the human breast cancer-derived cell line in a 96-well plate for three days and then transferred to a dish for fixation. As model agents, 1 mM ferrocenemethanol (FcCH2OH) and 1 mM potassium ferrocyanide ([Fe(CN)6]4-) were used as hydrophobic and hydrophilic substances, respectively. The microelectrode was inserted into the spheroid from the surface toward the center at 10 µm intervals, and cyclic voltammetry was performed at each position. The electrode was then fixed at the center (approximately 150 µm depth), and chronoamperometry was conducted after the addition of each agent to a final concentration of 1 mM.
[Results]The current decreased as the electrode approached the center of the spheroid, indicating limited diffusion into the inner region. The current of the hydrophilic agent decreased to 9.6% at the center, whereas that of the hydrophobic agent remained at 36.4%, demonstrating a clear difference in permeability. Differences were also observed in the time required for the current to reach a steady state after the addition of each agent. The hydrophobic agent exhibited a slower rise and required a longer time to stabilize.
[Consideration]The hydrophilic and negatively charged Fe(CN)6]4- cannot permeate the cell membrane and mainly diffuses through intercellular spaces, which limits its transport to the spheroid center. In contrast, hydrophobic FcCH2OH can permeate the cell membrane and diffuse via both intracellular and extracellular pathways, resulting in higher accessibility to the inner region. Differences in the rise time and stabilization of the current are attributed to hydrophobic interactions between FcCH2OH and cellular components such as lipid membranes, which transiently retain the molecules within the tissue and delay transport.
[Conclusion]In this study, the diffusion behavior of molecules within three-dimensional cellular structures was evaluated in spatial and temporal aspects. This method is expected to be applicable to the evaluation of the penetration behavior of drugs and chemical components in daily-use products.
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