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.
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。