PDF(Pubmed)
Abstract：
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
摘要：
背景：年龄相关性黄斑变性（AMD）是一种常见的眼部病理，主要影响老年人群。AMD的特征是进行性视网膜色素上皮（RPE）细胞变性，主要由抗氧化防御受损引起。AMD的治疗方法之一是将健康的RPE细胞注入视网膜下空间，需要纯净，健康的RPE细胞悬浮液。这项研究旨在电表征RPE细胞，以证明使用模拟通过介电电泳将健康RPE细胞与健康/氧化细胞的混合物分离的可能性。
方法：将BPEI-1大鼠RPE细胞暴露于过氧化氢以创建体外AMD细胞模型。使用各种方法评估细胞活力，包括显微成像，基于阻抗的实时细胞分析，和MTS测定。健康和氧化的细胞通过记录它们的介电泳光谱来表征，和电池参数(交叉频率，膜电导率和介电常数，和细胞质电导率）进行计算。使用这些参数在理论的基于微流体的介电泳分离芯片上进行了COMSOL模拟。
结果：增加过氧化氢浓度使第一个交叉频率向较低值移动，细胞膜介电常数逐渐增加。这些变化归因于进行性膜过氧化，当在用抗氧化剂N-乙酰半胱氨酸处理的细胞上测量时，它们减少了。交叉频率的变化足以有效分离健康细胞，如模拟所示。
结论：该研究表明，介电电泳可用于根据其电特性将健康的RPE细胞与氧化的RPE细胞分离。这种方法可能是一种可行的方法，用于AMD治疗程序的健康RPE细胞悬浮液。
方法：将BPEI-1大鼠RPE细胞暴露于过氧化氢以创建体外AMD细胞模型。使用各种方法评估细胞活力，包括显微成像，基于阻抗的实时细胞分析，和MTS测定。健康和氧化的细胞通过记录它们的介电泳光谱来表征，和电池参数(交叉频率，膜电导率和介电常数，和细胞质电导率）进行计算。使用这些参数在理论的基于微流体的介电泳分离芯片上进行了COMSOL模拟。
结果：增加过氧化氢浓度使第一个交叉频率向较低值移动，细胞膜介电常数逐渐增加。这些变化归因于进行性膜过氧化，当在用抗氧化剂N-乙酰半胱氨酸处理的细胞上测量时，它们减少了。交叉频率的变化足以有效分离健康细胞，如模拟所示。
结论：该研究表明，介电电泳可用于根据其电特性将健康的RPE细胞与氧化的RPE细胞分离。这种方法可能是一种可行的方法，用于AMD治疗程序的健康RPE细胞悬浮液。
