Assistant Professor National University of Singapore, United States
Introduction:: Stem cells are key in homeostasis and regeneration of tissues. Despite being in their own specialized niches, many stem cells move out and migrate long distances through complex architectures and microenvironments to accomplish their functions. During this journey it experiences different kinds of compressive and confining forces which dictates its cell fate. We have shown that when adult stem cells undergo confined migration through microfluidic chips, they differentiate into different cell types based on the degree of confinement. However, this approach is low throughput, allowing for the observation of 10s to 100s of cells per experiment.
The mechanical properties of the individual building blocks of granular systems can be controlled in terms of stiffness, curvature, and the degree of volumetric confinement they exert on the cells. Mechanical cues like confinement, curvature, stiffness and 3D microenvironments have been shown to regulate stem cell differentiation. Divergent differentiation of MSCs can be obtained using size dependent microgel gradients, even though it is unclear how this outcome is achieved. Moreover, 3D geometry, specifically the surface curvature of substrates, can induce mesenchymal stem cells to differentiate into osteogenic cells, dictate their migration speed and mode.
We have built a high throughput granular confinement system that allow us to study complex confinement-based cell behaviors by imposing confinement onto millions of cells in a single well before isolating mechanically primed cell populations for downstream characterization. Volumetric 3D confinement of cells will allow for the exploration of stem cell proliferation, fate, and death as a function of confinement.
Materials and Methods:: To create our 3D high throughput granular confinement system, glass spheres of 30-50 µm diameter were used as building blocks. Migratory and highly proliferative MDA-MB-231 breast cancer cells were attached to these glass spheres over a span of 24 hours, after which the spheres and their attached cells were placed into a cylindrical PDMS holder of diameter 3 mm and height 3 mm. The sphere-cell mixture then settled into a randomly packed aggregate, resulting in a homogeneous 3D void space with dimensions between 9-15 µm through which cells could migrate. After allowing cells to experience this volumetric confinement for 24, 48, and 72 hours, they were detached from the spheres using trypsin and replated onto 2D surfaces for 2 hours, then fixed and stained to visualize actin, the nucleus, and YAP localization. Morphological changes caused due to the mechanical treatment of these cells were then characterized by comparing with cells grown only on beads (2.5D) and glass substrates (2D).
Results, Conclusions, and Discussions:: We successfully fabricated granular systems composed of glass microspheres of multiple diameters. Using fluorescently labelled media, we characterized the void space through which cells could migrate. Next, we optimized our plating protocols to find parameters yielding the highest number of cells undergoing 3D confinement, and successfully kept MDA-MB-231 cells alive for over 48 hours inside the granular system. We observed that after confinement in the 3D granular scaffold, cells exhibited reduced cell area at the 24 and 48 hour time points as compared to the 2.5D scaffold and the 2D flat surface control cells, suggesting that their time within the granular system conveyed a mechanical memory that persisted after passaging. Interestingly, the nuclear area showed a reverse trend, increasing in area after confinement for 24 hours, revealing that cellular and nuclear adaptation to confinement may be distinct processes. When replating time was extended from 2 hours to 24 hours, no differences were observed between the control and experimental groups, as cells may be losing the mechanical memory of confinement. Stem cells were also plated inside these granular systems and assayed for differentiation at multiple time points to better understand the dynamics of fate specification within complex microenvironments. In both cancer cells and stem cells, we analysed the expression and localization of YAP, Lamin, and other mechanosensitive proteins via immunofluorescence and qPCR. Overall, understanding cell behaviour in 3D granular systems providing specific levels of confinement will lead to better control of cellular outputs, an enhanced understanding of confinement mechanobiology, and new tools for future tissue engineering approaches.
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