一种新型的用于研究电化疗疗效的Lab-On-A芯片微器件

IntroductionLab-on-a-Chip systems are innovative tools which can be used in the field of life sciences. They find applications e.g. in single cell analysis or cytotoxicity tests [1]. Despite the growing popularity of the use of microsystems in biological research, there is a lack of microsystems that may be used in the electrochemotherapy (ECT) studies. ECT is a antitumor therapy, based on an application of electroporation (EP) during standard chemotherapy (CT). EP uses external electric field to form hydrophilic pores in the cells membrane. Electropores are additional migration pathway for molecules which enhanced their delivery into cells [2]. ECT allows to use lower concentration of drug and reduces side effects in comparison to standard chemotherapy [3]. We develop a Lab-on-a-Chip microsystem for cell electroporation that could be used to examine the effectiveness of chemotherapy as well as for evaluation the effectiveness of electrochemotherapy.Microsystem fabricationThe microsystem is made of polydimethylsiloxane (PDMS) and glass. Casting method was used to obtained the microchannels and microchambers in PDMS layer. Pairs of gold electrodes were arranged parallelly along the microchannel with microchambers at the distance of 2 mm. The microsystem allows simultaneous culturing of normal and tumor cells. There are four rows of microchambers for each cell line: I - cells not exposed to compound or electric field (control), II - electroporated cells not exposed to compound (control for EP), III – cells electroporated with compound (simulating condition of ECT), IV - cells incubated with compound (simulating condition of CT). In this way, it is possible to evaluate and compare the effectiveness of two types of therapeutic procedures. Method The microchip was sterilized using 70% ethanol and UV radiation. After that, the cell suspension was introduced using a peristaltic pump at a speed of 3.5 µl/min. After 24 h of incubation, the cells medium (control) and the solution of the test compound were respectively introduced into the microsystem, nextly the cells were electroporated. Cell observations were performed using an inverted fluorescence microscope. In addition, fluorescence intensity measurements of the introduced molecules were carried out using a multi-well plate reader. The AlamarBlue test was performed to determine cell viability. For this purpose, a 10% AlamarBlue solution was introduced into the microsystem, than fluorescence intensity was measured at λex=558 nm and λem= 585 nm. Results and Conclusions To determine the optimal electroporation parameters (pulse length, their number, voltage) preliminary experiments were led using propidium iodide (PI). Tests were carried out for two skin cell lines: normal HaCaT and tumor A375. Two sets of parameters were examined: 1 pulse 10 ms and 8 pulses 0.1 ms, each in three voltage variants: 150, 180 and 200V. Cell viability after electroporation was determined. It was found that there were no significant changes in the cell viability after electroporation with the voltage lower than 200V. The efficiency of PI delivery into cells was confirmed by microscopic observation as well as determined by fluorescence intensity measurements. Significantly lower PI level inside cells (at the level of 30%) using 8 pulses 0.1 ms for both cell lines was observed. The best efficiency of PI delivery (about 90%) was observed when 1 pulse of 180V lasting 10 ms was applied. In addition, cell morphology was observed and cells parameters such as: shape factor, sphericity, convexity and elongation were determined. It was confirmed that the electroporation of cells does not change their morphology. Based on the obtained results it was concluded that the optimal electroporation conditions for HaCaT and A375 cell lines are: 1 pulse 10 ms 180V. References [1] Grabowska-Jadach I., Haczyk M., Drozd M., Fischer A., Pietrzak M., Malinowska E., Brzózka Z., \"Evaluation of biological activity of quantum dots in a microsystem\