skip to main content


Title: Pneumatic Non-Equibiaxial Cell Stretching Device With Live-Cell Imaging
Objective: Adherent cell behavior is influ- enced by a complex interplay of factors, including chemical and mechanical signals. In vitro experiments that mimic the mechanical environment experienced by cells in vivo are crucial for understanding cellular behavior and the progression of disease. In this study, we developed and validated a low-cost pneumatically-controlled cell stretcher with independent control of strain in two directions of a membrane, enabling unequal biaxial stretching and real- time microscopy during actuation. Methods: The stretch- ing was achieved by two independent pneumatic channels controlled by electrical signals. We used finite element simulations to compute the membrane’s strain field and particle tracking algorithms based on image processing techniques to validate the strain fields and measure the cell orientation and morphology. Results: The device can supply uniaxial, equibiaxial, and unequal biaxial stretching up to 15% strain in each direction at a frequency of 1Hz, with a strain measurement error of less than 1%. Through live cell imaging, we determined that distinct stretching patterns elicited differing responses and alterations in cell orientation and morphology, particularly in terms of cell length and area. Conclusion: The device successfully pro- vides a large, uniform, and variable strain field for cell experiments, while also enabling real-time, live cell imag- ing. Significance: This scalable, low-cost platform provides mechanical stimulation to cell cultures by independently controlling strains in two directions. This could contribute to a deeper understanding of cellular response to bio- realistic strains and could be useful for future in vitro drug testing platforms.  more » « less
Award ID(s):
2149946
PAR ID:
10517195
Author(s) / Creator(s):
; ; ; ; ;
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
  1. Abstract

    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.

     
    more » « less
  2. ABSTRACT  
    more » « less
  3. 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
  4. 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 wafer

    Basic Protocol 2: Cell confinement assay

    Support Protocol 1: Fixation and staining of cells after confinement

    Support protocol 2: Live/dead staining of cells during confinement

     
    more » « less
  5. 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 geometriesin 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 constructs

    Basic Protocol 2: Polymerization of micropatterned, bead‐doped polyacrylamide gel on an elastomer membrane

    Support Protocol: Cell culture and seeding onto CμBS constructs

    Basic Protocol 3: Implementing CμBS stretching protocols and traction force microscopy

    Basic Protocol 4: Data analysis and cell stress measurements

     
    more » « less