Characterization of collagen gel solutions and collagen matrices for cell culture

Sheu, Ming Thau; Huang, Jianjun; Yeh, Geng Chang; Ho, Hsiu O.

doi:10.1016/s0142-9612(00)00315-x

Cited by 204 publications

(161 citation statements)

References 8 publications

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“…To enhance AFM resolution, gels were prepared as in 2.1. and fixed with 1% glutaraldehyde (Sigma) PBS solution for 30 min to render stiffer gels [24]. Samples were imaged …”

Section: Afm Imagingmentioning

confidence: 99%

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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“…To enhance AFM resolution, gels were prepared as in 2.1. and fixed with 1% glutaraldehyde (Sigma) PBS solution for 30 min to render stiffer gels [24]. Samples were imaged …”

Section: Afm Imagingmentioning

confidence: 99%

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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“…Cross-linkers such as resimene, 4 epichlorohydrin, 5 genipin 6 and glutaraldehyde 7 were reported for chitosan. Chromium, 8 aldehydes, 9 hexamethylene diisocyanate, 10 carbodiimide, 11 acylazides 12 citric acid, maleic acid derivatives 13 and various other physical treatments like UV 14 were reported for type-I collagen. All the above said exogenous cross-linkers, cross-linked with chitosan or with type-I collagen through; (i) covalent amide/imine linkage; (ii) metal-protein complex formation (chromium cross-linking with type-I collagen); (iii) H-bond formation (between polyphenolic -OH group with different type of amino acids of type-I collagen molecule and amino groups of chitosan) etc.…”

Section: Introductionmentioning

confidence: 99%

Studies on Cross-linking of succinic acid with chitosan/collagen

et al. 2013

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The present study summarizes the cross-linking property of succinic acid with chitosan /collagen. In detail, the chemistry behind the cross-linking and the improvement in mechanical and thermal properties of the cross-linked material were discussed with suitable instruments and bioinformatics tools. The concentration of succinic acid with reference to the chosen polymers was optimized. A 3D scaffold prepared using an optimized concentration of succinic acid (0.2% (w/v)) with chitosan (1.0% (w/v)) and similarly with collagen (0.5% (w/v)), was subjected to surface morphology, FT-IR analysis, tensile strength assessment, thermal stability and biocompatibility. Results revealed, cross-linking with succinic acid impart appreciable mechanical strength to the scaffold material. In silico analysis suggested the prevalence of non-covalent interactions, which played a crucial role in improving the mechanical and thermal properties of the cross-linked scaffold. The resultant 3D scaffold may find application as wound dressing material, as an implant in clinical applications and as a tissue engineering material.

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“…Although a number of crosslinkers have been used for increasing the mechanical properties of these gels, the majority of these agents are cytotoxic or may induce an immune response when constructs are implanted in vivo. [20][21][22] Non-enzymatic glycation, the crosslinking of proteins via reducing sugars, has been shown to change the mechanical properties of proteins both in vivo and in vitro, [23][24][25][26][27][28][29] and is a recognized method for in situ processing of cell-seeded tissue constructs. 23,30 This mechanism of crosslinking has been developed to process collagen gels both in their solid state and in solution.…”

mentioning

confidence: 99%

Non‐enzymatic glycation of chondrocyte‐seeded collagen gels for cartilage tissue engineering

Roy¹,

Boskey²,

Bonassar³

2008

Journal Orthopaedic Research

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Collagen glycated with ribose (250 mM) in solution (pre-glycation) and as a gel (post-glycation) was seeded with chondrocytes and the effects of glycation on chondrocyte matrix assembly in culture were determined. Pre-glycation enhanced GAG accumulation significantly over controls at both 2 and 4 weeks (p < 0.05), although at both time points there were no statistical differences in cell number between pre-glycated and control gels. The increased proteoglycan accumulation was shown to be in part due to significantly increased GAG retention by the pre-glycated constructs (p < 0.05). Total collagen content in these pre-glycated gels was also significantly higher than unglycated gels at 4 weeks (p < 0.05). With post-glycation of collagen gels, chondrocyte number and GAG accumulation were all significantly lower than controls (p < 0.05). Post-glycation also inhibited GAG retention by the constructs (p < 0.05). Given these results, pre-glycation may be an improved processing method for collagen gels for tissue engineering techniques. ß

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Characterization of collagen gel solutions and collagen matrices for cell culture

Cited by 204 publications

References 8 publications

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

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

Studies on Cross-linking of succinic acid with chitosan/collagen

Non‐enzymatic glycation of chondrocyte‐seeded collagen gels for cartilage tissue engineering

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