Influence of multi-axial dynamic constraint on cell alignment and contractility in engineered tissues

AbstractIn this study an experimental rig is developed to investigate the influence of tissue constraint and cyclic loading on cell alignment and active cell force generation in uniaxial and biaxial engineered tissues constructs. Addition of contractile cells to collagen hydrogels dramatically increases the measured forces in uniaxial and biaxial constructs under dynamic loading. This increase in measured force is due to active cell contractility, as is evident from the decreased force after treatment with cytochalasin-D. Prior to dynamic loading, cells are highly aligned in uniaxially constrained tissues but are uniformly distributed in biaxially constrained tissues, demonstrating the importance of tissue constraints on cell alignment. Dynamic uniaxial stretching resulted in a slight increase in cell alignment in the centre of the tissue, whereas dynamic biaxial stretching had no significant effect on cell alignment. Our active modelling framework accurately predicts our experimental trends and suggests that a slightly higher (3%) total SF formation occurs at the centre of a biaxial tissue compared to the uniaxial tissue. However, high alignment of SFs and lateral compaction in the case of the uniaxially constrained tissue results in a significantly higher (75%) actively generated cell contractile stress, compared to the biaxially constrained tissue. These findings have significant implications for engineering of contractile tissue constructs.

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Influence of multi-axial dynamic constraint on cell alignment and contractility in engineered tissues

Journal of the Mechanical Behavior of Biomedical Materials ◽

10.1016/j.jmbbm.2020.104024 ◽

2020 ◽

Vol 112 ◽

pp. 104024

Author(s):

Noel H. Reynolds ◽

Eoin McEvoy ◽

Juan Alberto Panadero Pérez ◽

Ryan J. Coleman ◽

J. Patrick McGarry

Keyword(s):

Cell Alignment ◽

Engineered Tissues ◽

Dynamic Constraint

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Rapid generation of collagen-based microtissues to study cell–matrix interactions

TECHNOLOGY ◽

10.1142/s2339547816400094 ◽

2016 ◽

Vol 04 (02) ◽

pp. 80-87 ◽

Cited By ~ 8

Author(s):

Marie-Elena Brett ◽

Alexandra L. Crampton ◽

David K. Wood

Keyword(s):

Large Scale ◽

Culture Conditions ◽

Encapsulated Cells ◽

Cell Contractility ◽

Tissue Constructs ◽

Cell Matrix ◽

Matrix Interactions ◽

Rapid Generation ◽

Cell Matrix Interactions

The objective of this study was to create a method for studying cell–matrix interactions in a physiologically relevant 3D protein-based tissue construct that could be scaled up to perform large-scale screens, study cell–matrix interactions on a population basis, or be remodeled by cells to build larger tissues. We have developed an easy-to-use method to miniaturize protein-based tissue constructs that maintains the 3D in vitro environment, while alleviating several obstacles associated with larger avascular tissue constructs. In this study, we demonstrate that (i) cells can interact with the 3D environment both while encapsulated or while interacting only with the surface of the microtissues, (ii) encapsulated cells are highly viable and, for the first time, (iii) microtissues on this size scale (~200 μm) can be used to quantify cell contractility. This versatile platform should facilitate large-scale screens in 3D in vitro culture conditions for drug development and high throughput mechanistic biology.

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Contractile dynamics change before morphological cues during fluorescence illumination

Scientific Reports ◽

10.1038/srep18513 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 6

Author(s):

S. G. Knoll ◽

W. W. Ahmed ◽

T. A. Saif

Keyword(s):

Morphological Changes ◽

Traction Force ◽

Live Cells ◽

Excitation Light ◽

Cell Contractility ◽

Force Dynamics ◽

Force Relaxation ◽

Morphological Cues ◽

Contractile Forces ◽

Cell Force

Abstract Illumination can have adverse effects on live cells. However, many experiments, e.g. traction force microscopy, rely on fluorescence microscopy. Current methods to assess undesired photo-induced cell changes rely on qualitative observation of changes in cell morphology. Here we utilize a quantitative technique to identify the effect of light on cell contractility prior to morphological changes. Fibroblasts were cultured on soft elastic hydrogels embedded with fluorescent beads. The adherent cells generated contractile forces that deform the substrate. Beads were used as fiducial markers to quantify the substrate deformation over time, which serves as a measure of cell force dynamics. We find that cells exposed to moderate fluorescence illumination (λ = 540–585 nm, I = 12.5 W/m2, duration = 60 s) exhibit rapid force relaxation. Strikingly, cells exhibit force relaxation after only 2 s of exposure, suggesting that photo-induced relaxation occurs nearly immediately. Evidence of photo-induced morphological changes were not observed for 15–30 min after illumination. Force relaxation and morphological changes were found to depend on wavelength and intensity of excitation light. This study demonstrates that changes in cell contractility reveal evidence of a photo-induced cell response long before any morphological cues.

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Engineering large-scale perfused tissues via synthetic 3D soft microfluidics

10.1101/2021.08.20.457148 ◽

2021 ◽

Author(s):

Sergei Grebeniuk ◽

Abdel Rahman Abdel Fattah ◽

Gregorius Rustandi ◽

Manoj Kumar ◽

Burak Toprakhisar ◽

...

Keyword(s):

Large Scale ◽

De Novo ◽

Small Scale ◽

Diffusion Limit ◽

Dense Networks ◽

Tissue Constructs ◽

Engineered Tissues ◽

Living Tissues

The vascularization of engineered tissues and organoids has remained a major unresolved challenge in regenerative medicine. While multiple approaches have been developed to vascularize in vitro tissues, it has thus far not been possible to generate sufficiently dense networks of small-scale vessels to perfuse large de novo tissues. Here, we achieve the perfusion of multi-mm3 tissue constructs by generating networks of synthetic capillary-scale 3D vessels. Our 3D soft microfluidic strategy is uniquely enabled by a 3D-printable 2-photon-polymerizable hydrogel formulation, which allows for precise microvessel printing at scales below the diffusion limit of living tissues. We demonstrate that these large-scale engineered tissues are viable, proliferative and exhibit complex morphogenesis during long-term in-vitro culture, while avoiding hypoxia and necrosis. We show by scRNAseq and immunohistochemistry that neural differentiation is significantly accelerated in perfused neural constructs. Additionally, we illustrate the versatility of this platform by demonstrating long-term perfusion of developing liver tissue. This fully synthetic vascularization platform opens the door to the generation of human tissue models at unprecedented scale and complexity.

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Complex structures from patterned cell sheets

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2015.0515 ◽

2017 ◽

Vol 372 (1720) ◽

pp. 20150515 ◽

Cited By ~ 10

Author(s):

M. Misra ◽

B. Audoly ◽

S. Y. Shvartsman

Keyword(s):

Computational Analysis ◽

Apical Cell ◽

Three Dimensional ◽

Complex Structures ◽

Vertex Model ◽

Cell Sheets ◽

Cell Contractility ◽

Modelling Framework ◽

Spatio Temporal ◽

Epithelial Sheets

The formation of three-dimensional structures from patterned epithelial sheets plays a key role in tissue morphogenesis. An important class of morphogenetic mechanisms relies on the spatio-temporal control of apical cell contractility, which can result in the localized bending of cell sheets and in-plane cell rearrangements. We have recently proposed a modified vertex model that can be used to systematically explore the connection between the two-dimensional patterns of cell properties and the emerging three-dimensional structures. Here we review the proposed modelling framework and illustrate it through the computational analysis of the vertex model that captures the salient features of the formation of the dorsal appendages during Drosophila oogenesis. This article is part of the themed issue ‘Systems morphodynamics: understanding the development of tissue hardware’.

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Entropic forces drive cellular contact guidance

10.1101/479071 ◽

2018 ◽

Author(s):

A.B.C. Buskermolen ◽

H. Suresh ◽

S.S. Shishvan ◽

A. Vigliotti ◽

A. DeSimone ◽

...

Keyword(s):

Cell Size ◽

Thermal Fluctuations ◽

Contact Guidance ◽

Cell Alignment ◽

Stress Fibre ◽

Modelling Framework ◽

Statistical Framework ◽

Wide Range ◽

Cellular Contact ◽

As Stress

AbstractContact guidance—the widely-known phenomenon of cell alignment induced by anisotropic environmental features—is an essential step in the organization of adherent cells, but the mechanisms by which cells achieve this orientational ordering remain unclear. Here we seeded myofibroblasts on substrates micropatterned with stripes of fibronectin and observed that contact guidance emerges at stripe widths much greater than the cell size. To understand the origins of this surprising observation, we combined morphometric analysis of cells and their subcellular components with a novel statistical framework for modelling non-thermal fluctuations of living cells. This modelling framework is shown to predict not only the trends but also the statistical variability of a wide range of biological observables including cell (and nucleus) shapes, sizes and orientations, as well as stress-fibre arrangements within the cells with remarkable fidelity. By comparing observations and theory, we identified two regimes of contact guidance: (i) guidance on stripe widths smaller than the cell size (w ≤ 160 μm), which is accompanied by biochemical changes within the cells, including increasing stress-fibre polarisation and cell elongation, and (ii) entropic guidance on larger stripe widths, which is governed by fluctuations in the cell morphology. Overall, our findings suggest an entropy-mediated mechanism for contact guidance associated with the tendency of cells to maximise their morphological entropy through shape fluctuations.

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Engineering large-scale perfused tissues via synthetic 3D soft microfluidics

10.21203/rs.3.rs-867063/v1 ◽

2021 ◽

Author(s):

Sergei Grebenyuk ◽

Abdel Rahman Abdel Fattah ◽

Gregorius Rustandi ◽

Manoj Kumar ◽

Burak Toprakhisar ◽

...

Keyword(s):

Large Scale ◽

De Novo ◽

Small Scale ◽

Diffusion Limit ◽

Dense Networks ◽

Tissue Constructs ◽

Engineered Tissues ◽

Living Tissues

Abstract The vascularization of engineered tissues and organoids has remained a major unresolved challenge in regenerative medicine. While multiple approaches have been developed to vascularize in vitro tissues, it has thus far not been possible to generate sufficiently dense networks of small-scale vessels to perfuse large de novo tissues. Here, we achieve the perfusion of multi-mm3 tissue constructs by generating networks of synthetic capillary-scale 3D vessels. Our 3D soft microfluidic strategy is uniquely enabled by a 3D-printable 2-photon-polymerizable hydrogel formulation, which allows for precise microvessel printing at scales below the diffusion limit of living tissues. We demonstrate that these large-scale engineered tissues are viable, proliferative and exhibit complex morphogenesis during long-term in-vitro culture, while avoiding hypoxia and necrosis. We show by scRNAseq and immunohistochemistry that neural differentiation is significantly accelerated in perfused neural constructs. Additionally, we illustrate the versatility of this platform by demonstrating long-term perfusion of developing liver tissue. This fully synthetic vascularization platform opens the door to the generation of human tissue models at unprecedented scale and complexity.

Download Full-text