A Novel Chemotaxis Assay in 3-D Collagen Gels by Time-Lapse Microscopy

Vasaturo, Angela; Caserta, Sergio; Russo, Ilaria; Preziosi, Valentina; Ciacci, Carolina; Guido, Stefano

doi:10.1371/journal.pone.0052251

Cited by 25 publications

(37 citation statements)

References 54 publications

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“…far before the equilibrium A c c e p t e d M a n u s c r i p t 16 regime. In support of this assumption, a recent study conducted with a chemotaxis assay similar to ours on COLI gels reported a difference between diffusivity data obtained with the semiinfinite model applied within the diffusion layer or with Finite-Element (F-E) analysis of 17% [15], which is very close to the ~15% discrepancy between our D and D FRAP values. The latter agreement suggests that the correction obtained with F-E analysis is modest, whereas it is much more computationally demanding.…”

Section: Discussionsupporting

confidence: 87%

“…Alternatively, a less challenging approach has been reported in chemotaxis assays in which diffusivity of fluorescently labelled tracers is assessed by time-lapse fluorescence intensity measurements by fluorescence microscopy. However, these chemotaxis assays are still limited in that they require customized sample holders [15,16].…”

Section: Page 4 Of 34mentioning

confidence: 99%

“…The simplest and most widely used strategy to solve Eq. (1) for the diffusion of an extended initial particle distribution C o assumes that the hydrogel is a semi-infinite slab, which corresponds to , , and [5,11,15], thereby eliciting [19] (2)…”

Section: Spectrophotometer-based Diffusivity Measurementsmentioning

confidence: 99%

“…Eq. (2) is a suitable approximation to model the diffusion within a finite-sized material when the thickness of the diffusion layer δ during the experimental time-window is smaller than that of the material, where δ can be assessed as [15,20].…”

Section: Spectrophotometer-based Diffusivity Measurementsmentioning

confidence: 99%

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A spectrophotometer-based diffusivity assay reveals that diffusion hindrance of small molecules in extracellular matrix gels used in 3D cultures is dominated by viscous effects

Galgoczy

Pastor

Colom

et al. 2014

Colloids and Surfaces B: Biointerfaces

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Please cite this article as: R. Galgoczy, I. Pastor, A. Colom, A. Giménez, F. Mas, J. Alcaraz, A spectrophotometer-based diffusivity assay reveals that diffusion hindrance of small molecules in extracellular matrix gels used in 3D cultures is dominated by viscous effects, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10. 1016/j.colsurfb.2014.05.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AbstractThe design of 3D culture studies remains challenging due to the limited understanding of extracellular matrix (ECM)-dependent hindered diffusion and the lack of simple diffusivity assays. To address these limitations, we set up a cost-effective diffusivity assay based on a Transwell plate and the spectrophotometer of a Microplate Reader, which are readily accessible to cell biology groups. The spectrophotometer-based assay was used to assess the apparent diffusivity D of FITC-dextrans with molecular weight (4-70 kDa) spanning the physiological range of signaling factors in a panel of acellular ECM gels including Matrigel, fibrin and type I collagen. Despite their technical differences, D data exhibited ~15% relative difference with respect to FRAP measurements. Our results revealed that diffusion hindrance of small particles is controlled by the enhanced viscosity of the ECM gel in conformance with the Stokes-Einstein equation rather than by geometrical factors. Moreover, we provided a strong rationale that the enhanced ECM viscosity is largely contributed to by unassembled ECM macromolecules. We also reported that gels with the lowest D exhibited diffusion hindrance closest to the large physiologic hindrance of brain tissue, which has a typical pore size much smaller than ECM gels.Conversely, sparse gels (≤ 1 mg/ml), which are extensively used in 3D cultures, failed to reproduce the hindered diffusion of tissues, thereby supporting that dense (but not sparse) ECM gels are suitable tissue surrogates in terms of macromolecular transport. Finally, the consequences of reduced diffusivity in terms of optimizing the design of 3D culture experiments were addressed in detail.

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Section: Discussionsupporting

confidence: 87%

Section: Page 4 Of 34mentioning

confidence: 99%

Section: Spectrophotometer-based Diffusivity Measurementsmentioning

confidence: 99%

Section: Spectrophotometer-based Diffusivity Measurementsmentioning

confidence: 99%

See 2 more Smart Citations

A spectrophotometer-based diffusivity assay reveals that diffusion hindrance of small molecules in extracellular matrix gels used in 3D cultures is dominated by viscous effects

Galgoczy

Pastor

Colom

et al. 2014

Colloids and Surfaces B: Biointerfaces

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show abstract

“…For the molecular transport we chose to simulate the diffusion of a 10 kDa molecule with a diffusion coefficient of 2 Â 10 À6 cm 2 /s in collagen gel. 16 We assumed that the chemical concentration within the two side channels is always reported in terms of normalized values, thus varying from 0 to 1. The density and the dynamic viscosity of the fluid in the side channels were 1000 kg/m 3 and 10 À3 kg/m/s, respectively.…”

Section: Methodsmentioning

confidence: 99%

Optimizing design and fabrication of microfluidic devices for cell cultures: An effective approach to control cell microenvironment in three dimensions

et al. 2014

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The effects of gradients of bioactive molecules on the cell microenvironment are crucial in several biological processes, such as chemotaxis, angiogenesis, and tumor progression. The elucidation of the basic mechanisms regulating cell responses to gradients requires a tight control of the spatio-temporal features of such gradients. Microfluidics integrating 3D gels are useful tools to fulfill this requirement. However, even tiny flaws in the design or in the fabrication process may severely impair microenvironmental control, thus leading to inconsistent results. Here, we report a sequence of actions aimed at the design and fabrication of a reliable and robust microfluidic device integrated with collagen gel for cell culturing in 3D, subjected to a predetermined gradient of biomolecular signals. In particular, we developed a simple and effective solution to the frequently occurring technical problems of gas bubble formation and 3D matrix collapsing or detaching from the walls. The device here proposed, in Polydimethylsiloxane, was designed to improve the stability of the cell-laden hydrogel, where bubble deprived conditioning media flow laterally to the gel. We report the correct procedure to fill the device with the cell populated gel avoiding the entrapment of gas bubbles, yet maintaining cell viability. Numerical simulations and experiments with fluorescent probes demonstrated the establishment and stability of a concentration gradient across the gel. Finally, chemotaxis experiments of human Mesenchymal Stem Cells under the effects of Bone Morphogenetic Protein-2 gradients were performed in order to demonstrate the efficacy of the system in controlling cell microenvironment. The proposed procedure is sufficiently versatile and simple to be used also for different device geometries or experimental setups

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