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Fibroblast viability and phenotypic changes within glycated stiffened three-dimensional collagen matrices.

Vicens-Zygmunt V, Estany S, Colom A, Montes-Worboys A, Machahua C, Sanabria AJ, Llatjos R, Escobar I, Manresa F, Dorca J, Navajas D, Alcaraz J, Molina-Molina M - Respir. Res. (2015)

Bottom Line: A promising approach is based on three-dimensional collagen type I matrices that are stiffened by cross-linking through non-enzymatic glycation with reducing sugars.Finally, a progressive contractile phenotype cell differentiation was associated with the contraction of these gels.The use of non-enzymatic glycation with a low ribose concentration may provide a suitable model with a mechanic and oxidative modified environment with cells embedded in it, which allowed cell proliferation and induced fibroblast phenotypic changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pneumology, Unit of Interstitial Lung Diseases, University Hospital of Bellvitge, Barcelona, Spain. vvicens@hotmail.com.

ABSTRACT

Background: There is growing interest in the development of cell culture assays that enable the rigidity of the extracellular matrix to be increased. A promising approach is based on three-dimensional collagen type I matrices that are stiffened by cross-linking through non-enzymatic glycation with reducing sugars.

Methods: The present study evaluated the biomechanical changes in the non-enzymatically glycated type I collagen matrices, including collagen organization, the advanced glycation end products formation and stiffness achievement. Gels were glycated with ribose at different concentrations (0, 5, 15, 30 and 240 mM). The viability and the phenotypic changes of primary human lung fibroblasts cultured within the non-enzymatically glycated gels were also evaluated along three consecutive weeks. Statistical tests used for data analyze were Mann-Whitney U, Kruskal Wallis, Student's t-test, two-way ANOVA, multivariate ANOVA, linear regression test and mixed linear model.

Results: Our findings indicated that the process of collagen glycation increases the stiffness of the matrices and generates advanced glycation end products in a ribose concentration-dependent manner. Furthermore, we identified optimal ribose concentrations and media conditions for cell viability and growth within the glycated matrices. The microenvironment of this collagen based three-dimensional culture induces α-smooth muscle actin and tenascin-C fibroblast protein expression. Finally, a progressive contractile phenotype cell differentiation was associated with the contraction of these gels.

Conclusions: The use of non-enzymatic glycation with a low ribose concentration may provide a suitable model with a mechanic and oxidative modified environment with cells embedded in it, which allowed cell proliferation and induced fibroblast phenotypic changes. Such culture model could be appropriate for investigations of the behavior and phenotypic changes in cells that occur during lung fibrosis as well as for testing different antifibrotic therapies in vitro.

No MeSH data available.


Related in: MedlinePlus

Post-glycated matrices support fibroblast viability for long periods. Primary human lung fibroblasts cultured within different glycated collagen DMEM matrices over a period of 21 days. a. Fluorescence determination using alamarBlue assay. b. LIVE/DEAD viability/cytotoxicity stained cells observed using confocal reflection microscopy. The viable cells are stained green, and the non-viable cells are stained red. a and b. Cell death occurred under all conditions at the highest ribose (R) concentration (240 mM), independently of the use of serum (FBS). The use of FBS increased the proliferation rate under the rest of the conditions (p < 0.01). a1 and b1 (0 % FBS). No cell proliferation was observed, but cells were alive during the 21 days. a2 and b2 (1 % FBS). A gradual increase in cell proliferation was observed under all conditions except R 240 mM, but the rates for R 5 mM (p < 0.05) and the control matrices (p < 0.01) were significantly different only between the 7th and 21st days. a3 and b3 (10 % FBS). An improved proliferation rate was observed with the use of 10 % of serum, which was significantly for the control (non-glycated) only between the 7th and 14th days (p < 0.05). Gel contraction was observed in ≤15 mM R conditions between the 14th and the 21st day. No gel contraction was observed at 30 mM R (explanation in discussion). It was not possible to determine the fluorescence in the controls and in matrices glycated using 5 mM R at the 21st day because of gel contraction and cells growing on the bottom of the well (a3). The scale bars correspond to 200 μm. The experiments were repeated three times, with similar results obtained
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Fig4: Post-glycated matrices support fibroblast viability for long periods. Primary human lung fibroblasts cultured within different glycated collagen DMEM matrices over a period of 21 days. a. Fluorescence determination using alamarBlue assay. b. LIVE/DEAD viability/cytotoxicity stained cells observed using confocal reflection microscopy. The viable cells are stained green, and the non-viable cells are stained red. a and b. Cell death occurred under all conditions at the highest ribose (R) concentration (240 mM), independently of the use of serum (FBS). The use of FBS increased the proliferation rate under the rest of the conditions (p < 0.01). a1 and b1 (0 % FBS). No cell proliferation was observed, but cells were alive during the 21 days. a2 and b2 (1 % FBS). A gradual increase in cell proliferation was observed under all conditions except R 240 mM, but the rates for R 5 mM (p < 0.05) and the control matrices (p < 0.01) were significantly different only between the 7th and 21st days. a3 and b3 (10 % FBS). An improved proliferation rate was observed with the use of 10 % of serum, which was significantly for the control (non-glycated) only between the 7th and 14th days (p < 0.05). Gel contraction was observed in ≤15 mM R conditions between the 14th and the 21st day. No gel contraction was observed at 30 mM R (explanation in discussion). It was not possible to determine the fluorescence in the controls and in matrices glycated using 5 mM R at the 21st day because of gel contraction and cells growing on the bottom of the well (a3). The scale bars correspond to 200 μm. The experiments were repeated three times, with similar results obtained

Mentions: The fibroblast viability in the glycated 3D collagen matrices at different FBS concentrations was determined quantitatively using the AlamarBlue assay, which assesses the extent of mitochondrial activity via fluorescence (redox reaction, Fig. 4a), and confirmed qualitatively by differential staining of live and dead cells visualized using confocal microscopy (Fig. 4b).Fig. 4


Fibroblast viability and phenotypic changes within glycated stiffened three-dimensional collagen matrices.

Vicens-Zygmunt V, Estany S, Colom A, Montes-Worboys A, Machahua C, Sanabria AJ, Llatjos R, Escobar I, Manresa F, Dorca J, Navajas D, Alcaraz J, Molina-Molina M - Respir. Res. (2015)

Post-glycated matrices support fibroblast viability for long periods. Primary human lung fibroblasts cultured within different glycated collagen DMEM matrices over a period of 21 days. a. Fluorescence determination using alamarBlue assay. b. LIVE/DEAD viability/cytotoxicity stained cells observed using confocal reflection microscopy. The viable cells are stained green, and the non-viable cells are stained red. a and b. Cell death occurred under all conditions at the highest ribose (R) concentration (240 mM), independently of the use of serum (FBS). The use of FBS increased the proliferation rate under the rest of the conditions (p < 0.01). a1 and b1 (0 % FBS). No cell proliferation was observed, but cells were alive during the 21 days. a2 and b2 (1 % FBS). A gradual increase in cell proliferation was observed under all conditions except R 240 mM, but the rates for R 5 mM (p < 0.05) and the control matrices (p < 0.01) were significantly different only between the 7th and 21st days. a3 and b3 (10 % FBS). An improved proliferation rate was observed with the use of 10 % of serum, which was significantly for the control (non-glycated) only between the 7th and 14th days (p < 0.05). Gel contraction was observed in ≤15 mM R conditions between the 14th and the 21st day. No gel contraction was observed at 30 mM R (explanation in discussion). It was not possible to determine the fluorescence in the controls and in matrices glycated using 5 mM R at the 21st day because of gel contraction and cells growing on the bottom of the well (a3). The scale bars correspond to 200 μm. The experiments were repeated three times, with similar results obtained
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Fig4: Post-glycated matrices support fibroblast viability for long periods. Primary human lung fibroblasts cultured within different glycated collagen DMEM matrices over a period of 21 days. a. Fluorescence determination using alamarBlue assay. b. LIVE/DEAD viability/cytotoxicity stained cells observed using confocal reflection microscopy. The viable cells are stained green, and the non-viable cells are stained red. a and b. Cell death occurred under all conditions at the highest ribose (R) concentration (240 mM), independently of the use of serum (FBS). The use of FBS increased the proliferation rate under the rest of the conditions (p < 0.01). a1 and b1 (0 % FBS). No cell proliferation was observed, but cells were alive during the 21 days. a2 and b2 (1 % FBS). A gradual increase in cell proliferation was observed under all conditions except R 240 mM, but the rates for R 5 mM (p < 0.05) and the control matrices (p < 0.01) were significantly different only between the 7th and 21st days. a3 and b3 (10 % FBS). An improved proliferation rate was observed with the use of 10 % of serum, which was significantly for the control (non-glycated) only between the 7th and 14th days (p < 0.05). Gel contraction was observed in ≤15 mM R conditions between the 14th and the 21st day. No gel contraction was observed at 30 mM R (explanation in discussion). It was not possible to determine the fluorescence in the controls and in matrices glycated using 5 mM R at the 21st day because of gel contraction and cells growing on the bottom of the well (a3). The scale bars correspond to 200 μm. The experiments were repeated three times, with similar results obtained
Mentions: The fibroblast viability in the glycated 3D collagen matrices at different FBS concentrations was determined quantitatively using the AlamarBlue assay, which assesses the extent of mitochondrial activity via fluorescence (redox reaction, Fig. 4a), and confirmed qualitatively by differential staining of live and dead cells visualized using confocal microscopy (Fig. 4b).Fig. 4

Bottom Line: A promising approach is based on three-dimensional collagen type I matrices that are stiffened by cross-linking through non-enzymatic glycation with reducing sugars.Finally, a progressive contractile phenotype cell differentiation was associated with the contraction of these gels.The use of non-enzymatic glycation with a low ribose concentration may provide a suitable model with a mechanic and oxidative modified environment with cells embedded in it, which allowed cell proliferation and induced fibroblast phenotypic changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pneumology, Unit of Interstitial Lung Diseases, University Hospital of Bellvitge, Barcelona, Spain. vvicens@hotmail.com.

ABSTRACT

Background: There is growing interest in the development of cell culture assays that enable the rigidity of the extracellular matrix to be increased. A promising approach is based on three-dimensional collagen type I matrices that are stiffened by cross-linking through non-enzymatic glycation with reducing sugars.

Methods: The present study evaluated the biomechanical changes in the non-enzymatically glycated type I collagen matrices, including collagen organization, the advanced glycation end products formation and stiffness achievement. Gels were glycated with ribose at different concentrations (0, 5, 15, 30 and 240 mM). The viability and the phenotypic changes of primary human lung fibroblasts cultured within the non-enzymatically glycated gels were also evaluated along three consecutive weeks. Statistical tests used for data analyze were Mann-Whitney U, Kruskal Wallis, Student's t-test, two-way ANOVA, multivariate ANOVA, linear regression test and mixed linear model.

Results: Our findings indicated that the process of collagen glycation increases the stiffness of the matrices and generates advanced glycation end products in a ribose concentration-dependent manner. Furthermore, we identified optimal ribose concentrations and media conditions for cell viability and growth within the glycated matrices. The microenvironment of this collagen based three-dimensional culture induces α-smooth muscle actin and tenascin-C fibroblast protein expression. Finally, a progressive contractile phenotype cell differentiation was associated with the contraction of these gels.

Conclusions: The use of non-enzymatic glycation with a low ribose concentration may provide a suitable model with a mechanic and oxidative modified environment with cells embedded in it, which allowed cell proliferation and induced fibroblast phenotypic changes. Such culture model could be appropriate for investigations of the behavior and phenotypic changes in cells that occur during lung fibrosis as well as for testing different antifibrotic therapies in vitro.

No MeSH data available.


Related in: MedlinePlus