Accurately replicating and analyzing cellular responses to mechanical cues is vital for exploring metastatic disease progression. However, many of the existing in vitro platforms for applying mechanical stimulation seed cells on synthetic substrates. To better recapitulate physiological conditions, a novel actuating platform is developed with the ability to apply tensile strain on cells at various amplitudes and frequencies in a high‐throughput multi‐well culture plate using a physiologically relevant substrate. Suspending fibrillar fibronectin across the body of the magnetic actuator provides a matrix representative of early metastasis for 3D cell culture that is not reliant on a synthetic substrate. This platform enables the culturing and analysis of various cell types in an environment that mimics the dynamic stretching of lung tissue during normal respiration. Metabolic activity, YAP activation, and morphology of breast cancer cells are analyzed within one week of cyclic stretching or static culture. Further, matrix degradation is significantly reduced in breast cancer cell lines with metastatic potential after actuation. These new findings demonstrate a clear suppressive cellular response due to cyclic stretching that has implications for a mechanical role in the dormancy and reactivation of disseminated breast cancer cells to macrometastases.
- Award ID(s):
- 2149946
- PAR ID:
- 10517195
- Publisher / Repository:
- IEEE Transactions on Biomedical Engineering
- Date Published:
- Journal Name:
- IEEE Transactions on Biomedical Engineering
- ISSN:
- 0018-9294
- Page Range / eLocation ID:
- 1 to 11
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
Abstract -
ABSTRACT Mechanical stretching and topographical cues are both effective mechanical stimulations for regulating cell morphology, orientation, and behaviors. The competition of these two mechanical stimulations remains largely underexplored. Previous studies have suggested that a small cyclic mechanical strain is not able to reorient cells that have been pre‐aligned by relatively large linear microstructures, but can reorient those pre‐aligned by small linear micro/nanostructures if the characteristic dimension of these structures is below a certain threshold. Likewise, for micro/nanostructures with a given characteristic dimension, the strain must exceed a certain magnitude to overrule the topographic cues. There are however no in‐depth investigations of such “thresholds” due to the lack of close examination of dynamic cell orientation during and shortly after the mechanical loading. In this study, the time‐dependent combinatory effects of active and passive mechanical stimulations on cell orientation are investigated by developing a micromechanical stimulator. The results show that the cells pre‐aligned by linear micro/nanostructures can be altered by cyclic in‐plane strain, regardless of the structure size. During the loading, the micro/nanostructures can resist the reorientation effects by cyclic in‐plane strain while the resistive capability (measured by the mean orientation angle change and the reorientation speed) increases with the increasing characteristic dimension. The micro/nanostructures also can recover the cell orientation after the cessation of cyclic in‐plane strain, while the recovering capability increases with the characteristic dimension. The previously observed thresholds are largely dependent on the observation time points. In order to accurately evaluate the combinatory effects of the two mechanical stimulations, observations during the active loading with a short time interval or endpoint observations shortly after the loading are preferred. This study provides a microengineering solution to investigate the time‐dependent combinatory effects of the active and passive mechanical stimulations and is expected to enhance our understanding of cell responses to complex mechanical environments. Biotechnol. Bioeng. 2016;113: 2191–2201. © 2016 Wiley Periodicals, Inc.
-
The heart has a dynamic mechanical environment contributed by its unique cellular composition and the resultant complex tissue structure. In pathological heart tissue, both the mechanics and cell composition can change and influence each other. As a result, the interplay between the cell phenotype and mechanical stimulation needs to be considered to understand the biophysical cell interactions and organization in healthy and diseased myocardium. In this work, we hypothesized that the overall tissue organization is controlled by varying densities of cardiomyocytes and fibroblasts in the heart. In order to test this hypothesis, we utilized a combination of mechanical strain, co-cultures of different cell types, and inhibitory drugs that block intercellular junction formation. To accomplish this, an image analysis pipeline was developed to automatically measure cell type-specific organization relative to the stretch direction. The results indicated that cardiac cell type-specific densities influence the overall organization of heart tissue such that it is possible to model healthy and fibrotic heart tissue in vitro. This study provides insight into how to mimic the dynamic mechanical environment of the heart in engineered tissue as well as providing valuable information about the process of cardiac remodeling and repair in diseased hearts.more » « less
-
Abstract Cells in living tissues are exposed to substantial mechanical forces and constraints imposed by neighboring cells, the extracellular matrix, and external factors. Mechanical forces and physical confinement can drive various cellular responses, including changes in gene expression, cell growth, differentiation, and migration, all of which have important implications in physiological and pathological processes, such as immune cell migration or cancer metastasis. Previous studies have shown that nuclear deformation induced by 3D confinement promotes cell contractility but can also cause DNA damage and changes in chromatin organization, thereby motivating further studies in nuclear mechanobiology. In this protocol, we present a custom‐developed, easy‐to‐use, robust, and low‐cost approach to induce precisely defined physical confinement on cells using agarose pads with micropillars and externally applied weights. We validated the device by confirming nuclear deformation, changes in nuclear area, and cell viability after confinement. The device is suitable for short‐ and long‐term confinement studies and compatible with imaging of both live and fixed samples, thus presenting a versatile approach to studying the impact of 3D cell confinement and nuclear deformation on cellular function. This article contains detailed protocols for the fabrication and use of the confinement device, including live cell imaging and labeling of fixed cells for subsequent analysis. These protocols can be amended for specific applications. © 2023 Wiley Periodicals LLC.
Basic Protocol 1 : Design and fabrication of the confinement device waferBasic Protocol 2 : Cell confinement assaySupport Protocol 1 : Fixation and staining of cells after confinementSupport protocol 2 : Live/dead staining of cells during confinement -
Abstract Characterizing the mechanical properties of single cells is important for developing descriptive models of tissue mechanics and improving the understanding of mechanically driven cell processes. Standard methods for measuring single‐cell mechanical properties typically provide isotropic mechanical descriptions. However, many cells exhibit specialized geometries
in vivo , with anisotropic cytoskeletal architectures reflective of their function, and are exposed to dynamic multiaxial loads, raising the need for more complete descriptions of their anisotropic mechanical properties under complex deformations. Here, we describe the cellular microbiaxial stretching (CμBS) assay in which controlled deformations are applied to micropatterned cells while simultaneously measuring cell stress. CμBS utilizes a set of linear actuators to apply tensile or compressive, short‐ or long‐term deformations to cells micropatterned on a fluorescent bead‐doped polyacrylamide gel. Using traction force microscopy principles and the known geometry of the cell and the mechanical properties of the underlying gel, we calculate the stress within the cell to formulate stress‐strain curves that can be further used to create mechanical descriptions of the cells, such as strain energy density functions. © 2022 Wiley Periodicals LLC.Basic Protocol 1 : Assembly of CμBS stretching constructsBasic Protocol 2 : Polymerization of micropatterned, bead‐doped polyacrylamide gel on an elastomer membraneSupport Protocol : Cell culture and seeding onto CμBS constructsBasic Protocol 3 : Implementing CμBS stretching protocols and traction force microscopyBasic Protocol 4 : Data analysis and cell stress measurements