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

Abstract

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.

Oxygen diffusion and consumption in extracellular matrix gels: Implications for designing three‐dimensional cultures

“…The latter range was consistent with recent D G measurements on fibrin gels reported elsewhere, thereby supporting our data analysis in acellular gels. The small decrease of D G with respect to water has been traditionally attributed to the geometrical obstructions posed by the ECM scaffold, although nongeometrical factors may also be implicated …”

Elastic properties of hydrogels and decellularized tissue sections used in mechanobiology studies probed by atomic force microscopy

“…Thus, collagen‐I should be used when culturing mesenchymal cells, rBM should be the choice for epithelial or endothelial cells, whereas fibrin and hyaluronic acid should be used for cell types involved in repair and neurons, respectively (Griffith and Swartz, ; Lau et al, ; Mouw et al, ). Details on how to prepare 3D cultures based on the latter ECM components can be found elsewhere (Colom et al, ; Galgoczy et al, ; Wang et al, ). In brief, the ECM solution is first prepared at its desired concentration and kept in the liquid state at cold temperature, which is typically provided by keeping the ECM solution in an ice bucket.…”

“…A major advantage of mechanobiology assays based on increasing collagen‐I concentration is that they can mimic the increase in collagen deposition that occurs in different diseases including sclerosis, fibrosis, and solid tumors. However, interpreting the results obtained with these assays can be somewhat challenging (Trappmann and Chen, ), since an increase in ECM concentration implies an increase in both ligand density and gel stiffness as well as a decrease in the diffusivity of soluble signaling factors within the gel (Galgoczy et al, ). Thus, dissecting what cellular responses are attributed to gel stiffening alone requires often performing additional experiments with independent assays.…”

“…3D hydrogels based on native ECM components have provided important new insights on the mechanobiology of a variety of cell types in distinct physiopathological contexts. In addition, we have recently reported that ECM hydrogels are suitable tissue surrogates in terms of molecular diffusion and oxygen tension (Colom et al, ; Galgoczy et al, ). However, the currently attainable E values with these hydrogels cover only the low range (0.1–3 kPa) of physiologic values, thereby rendering them suboptimal to study the mechanobiology of tissues with intermediate or larger E values such as muscle, tendons, and bone (Butcher et al, ).…”

“…Details on these synthetic based assays have been reviewed elsewhere (Trappmann and Chen, ). However, these synthetic assays exhibit their own limitations, including porosity changes affecting the diffusion of soluble factors with variations in material stiffness (Galgoczy et al, ; Trappmann and Chen, ). Indeed, the development of 3D assays for mechanobiology research is a very active field of research.…”