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
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 sp...
| Autores: | , , , , , |
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| Tipo de recurso: | artículo |
| Estado: | Versión aceptada para publicación |
| Fecha de publicación: | 2014 |
| País: | España |
| Institución: | Varias* (Consorci de Biblioteques Universitáries de Catalunya, Centre de Serveis Científics i Acadèmics de Catalunya) |
| Repositorio: | Recercat. Dipósit de la Recerca de Catalunya |
| OAI Identifier: | oai:recercat.cat:2445/104308 |
| Acceso en línea: | https://hdl.handle.net/2445/104308 |
| Access Level: | acceso abierto |
| Palabra clave: | Matriu extracel·lular Cultiu cel·lular Extracellular matrix Cell culture |
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oai:recercat.cat:2445/104308 |
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España |
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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, Roland Pastor, Isabel Colom, Adai Giménez Hidalgo, Alicia Mas i Pujadas, Francesc Alcaraz Casademunt, Jordi Matriu extracel·lular Cultiu cel·lular Extracellular matrix Cell culture 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. Elsevier B.V. https://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 |
| spellingShingle |
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, Roland Matriu extracel·lular Cultiu cel·lular Extracellular matrix Cell culture |
| title_short |
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 |
| title_full |
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 |
| title_fullStr |
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 |
| title_full_unstemmed |
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 |
| title_sort |
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 |
| author_facet |
Galgoczy, Roland Pastor, Isabel Colom, Adai Giménez Hidalgo, Alicia Mas i Pujadas, Francesc Alcaraz Casademunt, Jordi |
| author_role |
author |
| author2 |
Pastor, Isabel Colom, Adai Giménez Hidalgo, Alicia Mas i Pujadas, Francesc Alcaraz Casademunt, Jordi |
| author2_role |
author author author author author |
| topic |
Matriu extracel·lular Cultiu cel·lular Extracellular matrix Cell culture |
| topic_facet |
Matriu extracel·lular Cultiu cel·lular Extracellular matrix Cell culture |
| description |
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. |
| publishDate |
2014 |
| format |
article |
| status_str |
acceptedVersion |
| url |
https://hdl.handle.net/2445/104308 |
| eu_rights_str_mv |
openAccess |
| publisher |
Elsevier B.V. |
| institution |
Varias* (Consorci de Biblioteques Universitáries de Catalunya, Centre de Serveis Científics i Acadèmics de Catalunya) |
| collection |
Recercat. Dipósit de la Recerca de Catalunya |
| reponame_str |
Recercat. Dipósit de la Recerca de Catalunya |
| instname_str |
Varias* (Consorci de Biblioteques Universitáries de Catalunya, Centre de Serveis Científics i Acadèmics de Catalunya) |
| _version_ |
1878740421118525440 |
| publishDateSort |
2014 |
| author_browse |
Alcaraz Casademunt, Jordi Colom, Adai Galgoczy, Roland Giménez Hidalgo, Alicia Mas i Pujadas, Francesc Pastor, Isabel |
| publisherStr |
Elsevier B.V. |
| score |
6,9008884 |