DEVELOPMENT OF A MICRO-WELL-BASED MICROFLUIDIC 3D CELL CULTURE FOR CYTOTOXICITY ASSAYS

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2023-1-26

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Farahani, Sarah

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This thesis presents a micro-well-based microfluidic platform's design, optimization, simulation, and fabrication for 3D cell culture. This work aims to create a system suitable for monitoring cytotoxicity assays on isolated chambers. In this context, microfluidic systems are promising for screening cell response to drugs. Here, a PMMA-based and pipette-operated microfluidic device for cytotoxicity assay is presented. The platform is incorporated into a channel and micro-chamber attached to the sidewall of the channel. The goal is to encapsulate two types of MCF-7 cellladen 3D collagen matrix namely as Red Fluorescent Protein (RFP)-MCF-7 cells and Wild-Type MCF-7 cells inside each well to form a 3D cell culture that can monitor the culture's response to different drug stimuli. This design is based on a level-set capillary flow of two multiphase flows, and the goal is to isolate the liquid inside a chamber. Based on the results obtained from Finite Element Analysis (FEA) simulations, a circle-shaped chamber, with a 2 mm diameter, placed at the intersection of the width of the rectangle-shaped channel, demonstrates the highest volume ratio of liquid confinement in the chamber. In the fabrication process, a micro-milling device is utilized to mill the structure on the PMMA substrate. The processed PMMA is sealed by surface-treatment-assisted thermo-compressive bonding of a blank PMMA sheet. The fabricated device is then tested by (RFP)-MCF-7 cell-laden 3D collagen matrix and Wild-Type MCF-7 cell-laden 3D collagen matrix. The cell suspension is at first pipetted through the inlet of the channel. As the mixture flows through the channel, it fills the chamber. Vacuuming the channel enables cell-laden collagen confinement in the chamber as well as chamber isolation. According to the observation of RFP-MCF-7 cell-laden 3D collagen matrix cultured in micro-wells, during a total of 48 hours of monitoring, the results show the success of cell growth in the wells in the first 24 hours of the experiment. The next 24 hours of the experiment were associated with the effect the of doxorubicin anticancer drug with 1µM and 10 µM concentrations on the cells, a dye-based live-dead assay was used to observe the viability of the cells during the experiment. Thus, Hoechst 33342 (blue fluorescence) and Propidium iodide (red fluorescence) were added to the suspension for the observation of live and dead cells, respectively. The corrected total cell fluorescence (CTCF) area was calculated for all control, 1µM treatment, and 10 µM treatment groups. Analyzing both fluorescent and Bright Field (BF) images of quantitative data regarding the effect of the treatment showed a decrease in the level of blue fluorescence and an increase in the intensity of dead cells as a red signal after 24 hours. Here, theoretical concepts, simulations of Finite Element Analysis (FEA), and the results of tests are discussed separately.

Subject Keywords

3D cell culture, microfluidics, drug research, cytotoxicity assays

URI

https://hdl.handle.net/11511/102777

Collections

Graduate School of Natural and Applied Sciences, Thesis

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Citation Formats

S. Farahani, “DEVELOPMENT OF A MICRO-WELL-BASED MICROFLUIDIC 3D CELL CULTURE FOR CYTOTOXICITY ASSAYS,” M.S. - Master of Science, Middle East Technical University, 2023.