Cell-seeded 3D scaffolds as in vitro models for electroporation.

Electroporation of cells is usually studied using cell suspensions or monolayer cultures. 3D scaffolds for cell culture have been recently designed in order to reproduce in vitro the complex and multifactorial environment experimented in vivo by cells. In fact, it is well known that 2D cell cultures are not able to simulate the complex interactions between the cells and their extracellular matrix (ECM). Recently, some examples of 3D models, like spheroids, have been investigated also in the electroporation field. Spheroids have been proposed in electrochemotherapy (ECT) studies to mimic tumor in vivo conditions: they are easy-to-handle 3D models but their sensitivity to electric field pulses depends from their diameter and, more interestingly, despite being relevant for intercellular junctions, they are not so much so for cell-ECM interactions. In this work, we propose a 3D macroscopic myxoid matrix for cell culture that would mimic the in vivo environment of myxoid stroma tumors. The myxoid stroma consists of abundant basic substances with large amounts of glycosaminoglycans (hyaluronic acid) and proteoglycans, poor collagen fibers and no elastin content. In the proposed approach, tumor cells seeded on 3D scaffolds mimic of myxoid stroma can establish both cell-cell and cell-ECM 3D interactions. The MCF7 cells (human breast adenocarcinoma cell line) were seeded in complete culture medium. Cell cultures were incubated at 37 °C for either 24 h, 3 days or 7 day. Some samples were used to assess cell vitality using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) test and others for electroporation tests and for histopathological analysis. The electroporation has been verified by the fluorescent dye Propidium cellular uptake. The proposed myxoid stroma scaffold induces cell proliferation and shows fibrous structures produced by cells, the concentration of which increases with culture time. The proposed matrix will be used for further investigations as a new scaffold for cell culture. Tumor cells grown into these new scaffolds will be used to evaluate electroporation including the stroma effect.

Effect of Electrode Distance in Grid Electrode: Numerical Models and In Vitro Tests.

Electrochemotherapy is an emerging local treatment for the management of superficial tumors and, among these, also chest wall recurrences from breast cancer. Generally, the treatment of this peculiar type of tumor requires the coverage of large skin areas. In these cases, electrochemotherapy treatment by means of standard small size needle electrodes (an array of 0.73 cm spaced needles, which covers an area of 1.5 cm2) is time-consuming and can allow an inhomogeneous coverage of the target area. We have previously designed grid devices suitable for treating an area ranging from 12 to 200 cm2. In this study, we propose different approaches to study advantages and drawbacks of a grid device with needles positioned 2 cm apart. The described approach includes a numerical evaluation to estimate electric field intensity, followed by an experimental quantification of electroporation on a cell culture. The electric field generated in a conductive medium has been studied by means of 3-dimensional numerical models with varying needle pair distance from 1 to 2 cm. In particular, the electric field evaluation shows that the electric field intensity with varying needle distance is comparable in the area in the middle of the 2 electrodes. Differently, near needles, the electric field intensity increases with the increasing electrode distance and supply voltage. The computational results have been correlated with experimental ones obtained in vitro on cell culture. In particular, electroporation effect has been assessed on human breast cancer cell line MCF7, cultured in monolayer. The use of 2-cm distant needles, supplied by 2000 V, produced an electroporation effect in the whole area comprised between the electrodes. Areas of cell culture where reversible and irreversible electroporation occurred were identified under microscope by using fluorescent dyes. The coupling of computation and experimental results could be helpful to evaluate the effect of the needle distance on the electric field intensity in cell cultures in terms of reversible or irreversible electroporation.