Browsing by Author "Phillippi, Julie A."

Now showing 1 - 3 of 3
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    Biophysical Influence of Nanofiber Networks to Direct Pericyte Aggregation into Spheroids
    Sharma, Sharan (Virginia Tech, 2023-07-25)
    Multicellular spheroids have emerged as a promising tool for drug delivery, cancer therapy, and tissue engineering. Compared to 2D monolayers, spheroids provide a more realistic representation of the 3D cellular environment, enabling better understanding of the signaling cascades and growth factors involved in vivo. The formation of in vitro spheroids involves the aggregation of several cells that proliferate to grow into larger spheroids. Biophysical cues provide crucial information for the cells to assemble into 3D structures. We used suspended fiber networks to demonstrate a new way to form and spatially pattern spheroids comprised of human pericytes. We show that fiber architecture (aligned vs. crosshatched), diameter (200, 500, and 800 nm), and contractility influence spheroids in their spontaneous formation, growth, and maintenance, and report a dynamic trade of cells between adjacent spheroids through remodeled fiber networks. We found that aligned fiber networks promoted spheroid formation independent of fiber diameter, while large-diameter crosshatched networks abrogated spheroid formation, promoting growth of 2D monolayers. Thus, a mixture of diameters and architectures allowed for spatial patterning of spheroids and monolayers within a single system. We further quantified various dynamic interactions and describe the forces involved during spheroid formation, cell efflux from spheroids, and show the loss and recovery of spheroid forces with pharmacological perturbation of Rho-associated protein kinase (ROCK). Thus, we develop new insights on the dynamics of spheroids using suspended fiber networks of varying diameters and architectures, with the potential to connect matrix biology with developmental, disease, and regenerative biology.
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    Cell-Fiber Interactions: A New Route to Mechano-Biological Investigations in Developmental and Disease Biology
    Sheets, Kevin Tyler (Virginia Tech, 2014-11-03)
    Cells in the body interact with a predominantly fibrous microenvironment and constantly adapt to changes in their neighboring physiochemical environment, which has implications in developmental and disease biology. A myriad of in vitro platforms including 2D flat and 3D gel substrates with and without anisotropy have demonstrated cellular alterations to subtle changes in topography. Recently, our work using suspended fibers as a new in vitro biological assay has revealed that cells are able to sense and respond to changes in fiber curvature and structural stiffness as evidenced by alterations to cytoskeleton arrangement, including focal adhesion cluster lengths and nucleus shape indices, leading to altered migration speeds. It is hypothesized that these behaviors occur due to modulation of cellular inside-out forces in response to changes in the external fibrous environment (outside-in). Thus, in this study, we investigate the role of fiber curvature and structural stiffness in force modulation of single cells attached to suspended fibers. Using our previously reported non-electrospinning Spinneret based Tunable Engineered Parameters (STEP) fiber manufacturing platform, we present our findings on single cell inside-out and outside-in forces using fibers of three diameters (250 nm, 400 nm and 800 nm) representing a wide range of structural stiffness (3-45 nN/μm). To investigate cellular adaptability to external perturbation, we present the development of a first-of-its-kind force measurement 'nanonet' platform capable of investigating cell adhesion forces in response to symmetric and non-symmetric (injury model) loading. Our combined findings are multi-fold: (i) Cells on suspended fibers are able to form focal adhesion clusters approximately four times longer than those on flat substrates, which gives them potential to double their migration speeds, (ii) Nanonets as force probes show that the contractility-based inside-out forces are nearly equally distributed on both sides of the cell body, and that overall force magnitudes are dependent on fiber structural stiffness, and (iii) External perturbation can evenly (symmetric) or unevenly (non-symmetric) distribute forces within the cell, and the resulting bias causes diameter-dependent outside-in adhesion force response. Finally, we demonstrate the power of the developed force measurement platform by extending our studies to cell-cell junctional forces as well as single-cell disease models including cancer and aortic aneurysm.
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    Nanonet force microscopy for measuring forces in single smooth muscle cells of the human aorta
    Hall, Alexander; Chan, Patrick; Sheets, Kevin; Apperson, Matthew; Delaughter, Christopher; Gleason, Thomas G.; Phillippi, Julie A.; Nain, Amrinder S. (2017-07-07)
    A number of innovative methods exist to measure cell-matrix adhesive forces, but they have yet to accurately describe and quantify the intricate interplay of a cell and its fibrous extracellular matrix (ECM). In cardiovascular pathologies, such as aortic aneurysm, new knowledge on the involvement of cell-matrix forces could lead to elucidation of disease mechanisms. To better understand this dynamics, we measured primary human aortic single smooth muscle cell (SMC) forces using nanonet force microscopy in both inside-out (I-O intrinsic contractility) and outside-in (O-I external perturbation) modes. For SMC populations, we measured the I-O and O-I forces to be 12.9 +/- 1.0 and 57.9 +/- 2.5 nN, respectively. Exposure of cells to oxidative stress conditions caused a force decrease of 57 and 48% in I-O and O-I modes, respectively, and an increase in migration rate by 2.5-fold. Finally, in O-I mode, we cyclically perturbed cells at constant strain of varying duration to simulate in vivo conditions of the cardiac cycle and found that I-O forces decrease with increasing duration and O-I forces decreased by half at shorter cycle times. Thus our findings highlight the need to study forces exerted and felt by cells simultaneously to comprehensively understand force modulation in cardiovascular disease.