Please use this identifier to cite or link to this item: http://hdl.handle.net/2445/104308
Title: 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
Author: Galgoczy, Roland
Pastor, Isabel
Colom, Adai
Giménez, Alicia
Mas i Pujadas, Francesc
Alcaraz Casademunt, Jordi
Keywords: Matriu extracel·lular
Cultiu cel·lular
Extracellular matrix
Cell culture
Issue Date: 2014
Publisher: Elsevier B.V.
Abstract: The 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 (4-70kDa) 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 (≤1mg/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.
Note: Versió postprint del document publicat a: https://doi.org/10.1016/j.colsurfb.2014.05.017
It is part of: Colloids and Surfaces B-Biointerfaces, 2014, vol. 120, p. 200-207
Related resource: https://doi.org/10.1016/j.colsurfb.2014.05.017
URI: http://hdl.handle.net/2445/104308
ISSN: 0927-7765
Appears in Collections:Articles publicats en revistes (Ciència dels Materials i Química Física)

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