Using 3D tissue fabrication to investigate epithelial-to-mesenchymal transition in cancer cell lines and trophoblast stem cells
Background
Prior research by the Huck group developed alginate hydrogels that support self-assembly of multicellular structures with a defined shape and size. These structures (microtissue) can be released from the alginate hydrogel to create freestanding microtissue (FMT). FMT can be embedded in fibrinogen gel. The stiffness of fibrinogen gel can be fine-tuned with relative ease. This enables us to study the migration of cells encompassing the microtissue in response to media with different stiffness. We will use immunostaining to localize cells that exhibit mesenchymal marker, vimentin, and epithelial marker, E-cadherin, to identify cells undergoing epithelial-to-mesenchymal transition (EMT). Cells predominantly expressing mesenchymal markers are known to exhibit increased motility and invasiveness, whereas cells expressing epithelial markers are non-motile. Therefore, we will use EMT to identify cells that have an increased capability to migrate. It is hypothesized that cells in the corners of the microtissue, where there is a high-stress environment, will exhibit a mesenchymal cell state.
Prior research by the Huck group developed alginate hydrogels that support self-assembly of multicellular structures with a defined shape and size. These structures (microtissue) can be released from the alginate hydrogel to create freestanding microtissue (FMT). FMT can be embedded in fibrinogen gel. The stiffness of fibrinogen gel can be fine-tuned with relative ease. This enables us to study the migration of cells encompassing the microtissue in response to media with different stiffness. We will use immunostaining to localize cells that exhibit mesenchymal marker, vimentin, and epithelial marker, E-cadherin, to identify cells undergoing epithelial-to-mesenchymal transition (EMT). Cells predominantly expressing mesenchymal markers are known to exhibit increased motility and invasiveness, whereas cells expressing epithelial markers are non-motile. Therefore, we will use EMT to identify cells that have an increased capability to migrate. It is hypothesized that cells in the corners of the microtissue, where there is a high-stress environment, will exhibit a mesenchymal cell state.
Objective
Two objectives for this research are defined. The first objective is to embed freestanding microtissue in fibrinogen gel with varying stiffness to examine the effect of environmental cues on cell migration. The second objective is to investigate whether confinement by a 3D microenvironment is a unique cue that induces EMT in trophoblast stem cells (TSCs).
Two objectives for this research are defined. The first objective is to embed freestanding microtissue in fibrinogen gel with varying stiffness to examine the effect of environmental cues on cell migration. The second objective is to investigate whether confinement by a 3D microenvironment is a unique cue that induces EMT in trophoblast stem cells (TSCs).
Material and methods
HeLa, A549, and TSC microtissue was fabricated in an RGD-coupled alginate hydrogel. After microtissue fabrication, the microtissue was released using a buffer containing citric acid (releasing buffer). Subsequently, freestanding microtissue was embedded in fibrinogen gel with varying stiffness. After embedding, cells were fixed, stained with fluorescent antibodies, and analysed using laser confocal scanning microscopy (LCSM).
HeLa, A549, and TSC microtissue was fabricated in an RGD-coupled alginate hydrogel. After microtissue fabrication, the microtissue was released using a buffer containing citric acid (releasing buffer). Subsequently, freestanding microtissue was embedded in fibrinogen gel with varying stiffness. After embedding, cells were fixed, stained with fluorescent antibodies, and analysed using laser confocal scanning microscopy (LCSM).
Results
Results from the first objective have shown that fibrinogen with high stiffness caused triangular FMT to deform into a round shape and no cell migration was observed. Fibrinogen with low stiffness caused cells to disperse in the medium and the original FMT structure was completely lost. Complications that developed because of COVID19 caused a premature stop to the experiments. Furthermore, these results are based on a single experiment. Therefore, it is suggested to repeat the experiment. Results from the second objective have shown that the induction of EMT in TSCs is not a unique expression of a confined 3D microenvironment, but also shared by 2D culturing methods.
Results from the first objective have shown that fibrinogen with high stiffness caused triangular FMT to deform into a round shape and no cell migration was observed. Fibrinogen with low stiffness caused cells to disperse in the medium and the original FMT structure was completely lost. Complications that developed because of COVID19 caused a premature stop to the experiments. Furthermore, these results are based on a single experiment. Therefore, it is suggested to repeat the experiment. Results from the second objective have shown that the induction of EMT in TSCs is not a unique expression of a confined 3D microenvironment, but also shared by 2D culturing methods.