Biophysics and Cellular Biotechnology Department, Faculty of Medicine, Carol Davila University of Medicine and Pharmacy, 8 Eroii Sanitari blvd., Bucharest, 050474, Romania.
Excellence Center for Research in Biophysics and Cellular Biotechnology, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania.
BMC Ophthalmol. 2024 Aug 13;24(1):340. doi: 10.1186/s12886-024-03617-0.
Age-related macular degeneration (AMD) is a prevalent ocular pathology affecting mostly the elderly population. AMD is characterized by a progressive retinal pigment epithelial (RPE) cell degeneration, mainly caused by an impaired antioxidative defense. One of the AMD therapeutic procedures involves injecting healthy RPE cells into the subretinal space, necessitating pure, healthy RPE cell suspensions. This study aims to electrically characterize RPE cells to demonstrate a possibility using simulations to separate healthy RPE cells from a mixture of healthy/oxidized cells by dielectrophoresis.
BPEI-1 rat RPE cells were exposed to hydrogen peroxide to create an in-vitro AMD cellular model. Cell viability was evaluated using various methods, including microscopic imaging, impedance-based real-time cell analysis, and the MTS assay. Healthy and oxidized cells were characterized by recording their dielectrophoretic spectra, and electric cell parameters (crossover frequency, membrane conductivity and permittivity, and cytoplasm conductivity) were computed. A COMSOL simulation was performed on a theoretical microfluidic-based dielectrophoretic separation chip using these parameters.
Increasing the hydrogen peroxide concentration shifted the first crossover frequency toward lower values, and the cell membrane permittivity progressively increased. These changes were attributed to progressive membrane peroxidation, as they were diminished when measured on cells treated with the antioxidant N-acetylcysteine. The changes in the crossover frequency were sufficient for the efficient separation of healthy cells, as demonstrated by simulations.
The study demonstrates that dielectrophoresis can be used to separate healthy RPE cells from oxidized ones based on their electrical properties. This method could be a viable approach for obtaining pure, healthy RPE cell suspensions for AMD therapeutic procedures.
年龄相关性黄斑变性（AMD）是一种常见的眼部病理学疾病，主要影响老年人群体。AMD 的特征是视网膜色素上皮（RPE）细胞进行性退化，主要是由于抗氧化防御功能受损所致。AMD 的一种治疗方法包括将健康的 RPE 细胞注射到视网膜下腔，这需要纯净、健康的 RPE 细胞悬浮液。本研究旨在对 RPE 细胞进行电特性分析，以证明通过介电泳分离健康 RPE 细胞和混合的健康/氧化细胞的可能性。
将 BPEI-1 大鼠 RPE 细胞暴露于过氧化氢中，以建立体外 AMD 细胞模型。采用多种方法评估细胞活力，包括显微镜成像、基于阻抗的实时细胞分析和 MTS 测定法。通过记录介电泳谱和计算电细胞参数（交叉频率、膜电导率和介电常数以及细胞质电导率）来对健康和氧化细胞进行特征描述。使用这些参数在理论微流控介电泳分离芯片上对 COMSOL 模拟进行了执行。
随着过氧化氢浓度的增加，第一交叉频率向较低值移动，并且细胞膜介电常数逐渐增加。这些变化归因于膜过氧化的进行，因为在用抗氧化剂 N-乙酰半胱氨酸处理的细胞上测量时，这些变化会减小。交叉频率的变化足以有效分离健康细胞，这一点通过模拟得到了证明。
该研究表明，基于其电特性，介电泳可用于分离健康的 RPE 细胞和氧化的 RPE 细胞。这种方法可能是获得用于 AMD 治疗程序的纯净、健康的 RPE 细胞悬浮液的可行方法。
