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Producing Collagen Micro-stripes with Aligned Fibers for Cell Migration Assays.

ABSTRACT: Introduction:The orientation of collagen fibers in native tissues plays an important role in cell signaling and mediates the progression of tumor cells in breast cancer by a contact guidance mechanism. Understanding how migration of epithelial cells is directed by the alignment of collagen fibers requires in vitro assays with standardized orientations of collagen fibers. Methods:To address this issue, we produced micro-stripes with aligned collagen fibers using an easy-to-use and versatile approach based on the aspiration of a collagen solution within a microchannel. Glass coverslips were functionalized with a (3-aminopropyl)triethoxysilane/glutaraldehyde linkage to covalently anchor micro-stripes of aligned collagen fibers, whereas microchannels were functionalized with a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) nonionic triblock polymer to prevent adhesion of the collagen micro-stripes. Results:Using this strategy, microchannels can be peeled off to expose micro-stripes of aligned collagen fibers without affecting their mechanical integrity. We used time-lapse confocal reflection microscopy to characterize the polymerization kinetics of collagen networks for different concentrations and the orientation of collagen fibers as a function of the microchannel width. Our results indicate a non-linear concentration dependence of the area of fluorescence, suggesting that the architecture of collagen networks is sensitive to small changes in concentration. We show the possibility to influence the collagen fibril coverage by adjusting the concentration of the collagen solution. Conclusion:We applied this novel approach to study the migration of epithelial cells, demonstrating that collagen micro-stripes with aligned fibers represent a valuable in-vitro assay for studying cell contact guidance mechanisms.

SUBMITTER: Mohammed D

PROVIDER: S-EPMC6981333 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Producing Collagen Micro-stripes with Aligned Fibers for Cell Migration Assays.

Mohammed Danahe D Pardon Gaspard G Versaevel Marie M Bruyère Céline C Alaimo Laura L Luciano Marine M Vercruysse Eléonore E Pruitt Beth L BL Gabriele Sylvain S

Cellular and molecular bioengineering 20190925 1

<h4>Introduction</h4>The orientation of collagen fibers in native tissues plays an important role in cell signaling and mediates the progression of tumor cells in breast cancer by a contact guidance mechanism. Understanding how migration of epithelial cells is directed by the alignment of collagen fibers requires <i>in vitro</i> assays with standardized orientations of collagen fibers.<h4>Methods</h4>To address this issue, we produced micro-stripes with aligned collagen fibers using an easy-to-u ...[more]

PMID: 32030110

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Project description:It is well established that collagen alignment in the breast tumor microenvironment provides biophysical cues to drive disease progression. Numerous mechanistic studies have demonstrated that tumor cell behavior is driven by the architecture and stiffness of the collagen matrix. However, the mechanical properties within a 3D collagen microenvironment, particularly at the scale of the cell, remain poorly defined. To investigate cell-scale mechanical cues with respect to local collagen architecture, we employed a combination of intravital imaging of the mammary tumor microenvironment and a 3D collagen gel system with both acellular pNIPAAm microspheres and MDA-MB-231 breast carcinoma cells. Within the in vivo tumor microenvironment, the displacement of collagen fiber was identified in response to tumor cells migrating through the stromal matrix. To further investigate cell-scale stiffness in aligned fiber architectures and the propagation of cell-induced fiber deformations, precise control of collagen architecture was coupled with innovative methodology to measure mechanical properties of the collagen fiber network. This method revealed up to a 35-fold difference in directional cell-scale stiffness resulting from contraction against aligned fibers. Furthermore, the local anisotropy of the matrix dramatically altered the rate at which contractility-induced fiber displacements decayed over distance. Together, our results reveal mechanical properties in aligned matrices that provide dramatically different cues to the cell in perpendicular directions. These findings are supported by the mechanosensing behavior of tumor cells and have important implications for cell-cell communication within the tissue microenvironment. STATEMENT OF SIGNIFICANCE: It is widely appreciated that the architecture of the extracellular matrix impacts cellular behavior in normal and disease states. Numerous studies have determined the fundamental role of collagen matrix architecture on cellular mechanosensing, but effectively quantifying anisotropic mechanical properties of the collagen matrix at the cell-scale remains challenging. Here, we developed innovative methodology to discover that collagen alignment results in a 35-fold difference in cell-scale stiffness and alters contractile force transmission through the fiber network. Furthermore, we identified bias in cell response along the axis of alignment, where local stiffness is highest. Overall, our results define cell-scale stiffness and fiber deformations due to collagen architecture that may instruct cell communication within a broad range of tissue microenvironments.

Project description:Biofabrication is providing scientists and clinicians the ability to produce engineered tissues with desired shapes and gradients of composition and biological cues. Typical resolutions achieved with extrusion-based bioprinting are at the macroscopic level. However, for capturing the fibrillar nature of the extracellular matrix (ECM), it is necessary to arrange ECM components at smaller scales, down to the micron and the molecular level. Herein, we introduce a bioink containing the tyramine derivative of hyaluronan (HA; henceforth known as THA) and collagen (Col) type 1. In this bioink, similar to connective tissues, Col is present in the fibrillar form, and HA functions as a viscoelastic space filler. THA was enzymatically cross-linked under mild conditions allowing simultaneous Col fibrillogenesis, thus achieving a homogeneous distribution of Col fibrils within the viscoelastic HA-based matrix. The THA-Col composite displayed synergistic properties in terms of storage modulus and shear thinning, translating into good printability. Shear-induced alignment of the Col fibrils along the printing direction was achieved and quantified via immunofluorescence and second-harmonic generation. Cell-free and cell-laden constructs were printed and characterized, analyzing the influence of the controlled microscopic anisotropy on human bone marrow-derived mesenchymal stromal cell (hMSC) migration. Anisotropic HA-Col showed cell-instructive properties modulating hMSC adhesion, morphology, and migration from micropellets stimulated by the presence and the orientation of Col fibers. Actin filament staining showed that hMSCs embedded in aligned constructs displayed increased cytoskeleton alignment along the fibril direction. Based on gene expression of cartilage/bone markers and ECM production, hMSCs embedded in the isotropic bioink displayed chondrogenic differentiation comparable with standard pellet culture by means of proteoglycan production (safranin O staining and proteoglycan quantification). The possibility of printing matrix components with control over microscopic alignment brings biofabrication one step closer to capturing the complexity of native tissues.

Project description:Carcinoma cells frequently expand and invade from a confined lesion, or multicellular clusters, into and through the stroma on the path to metastasis, often with an efficiency dictated by the architecture and composition of the microenvironment. Specifically, in desmoplastic carcinomas such as those of the breast, aligned collagen tracks provide contact guidance cues for directed cancer cell invasion. Yet, the evolving dynamics of this process of invasion remains poorly understood, in part due to difficulties in continuously capturing both spatial and temporal heterogeneity and progression to invasion in experimental systems. Therefore, to study the local invasion process from cell dense clusters into aligned collagen architectures found in solid tumors, we developed a novel engineered 3D invasion platform that integrates an aligned collagen matrix with a cell dense tumor-like plug. Using multiphoton microscopy and quantitative analysis of cell motility, we track the invasion of cancer cells from cell-dense bulk clusters into the pre-aligned 3D matrix, and define the temporal evolution of the advancing invasion fronts over several days. This enables us to identify and probe cell dynamics in key regions of interest: behind, at, and beyond the edge of the invading lesion at distinct time points. Analysis of single cell migration identifies significant spatial heterogeneity in migration behavior between cells in the highly cell-dense region behind the leading edge of the invasion front and cells at and beyond the leading edge. Moreover, temporal variations in motility and directionality are also observed between cells within the cell-dense tumor-like plug and the leading invasive edge as its boundary extends into the anisotropic collagen over time. Furthermore, experimental results combined with mathematical modeling demonstrate that in addition to contact guidance, physical crowding of cells is a key regulating factor orchestrating variability in single cell migration during invasion into anisotropic ECM. Thus, our novel platform enables us to capture spatio-temporal dynamics of cell behavior behind, at, and beyond the invasive front and reveals heterogeneous, local interactions that lead to the emergence and maintenance of the advancing front.

Dataset Information

Producing Collagen Micro-stripes with Aligned Fibers for Cell Migration Assays.

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Producing Collagen Micro-stripes with Aligned Fibers for Cell Migration Assays.

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