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Measurement of biomolecular diffusion in extracellular matrix condensed by fibroblasts using fluorescence correlation spectroscopy.

Kihara T, Ito J, Miyake J - PLoS ONE (2013)

Bottom Line: Additionally, the diffusion coefficients of biomolecules were inversely correlated with molecular radius.Furthermore, molecular diffusion was greatly restricted, with a central focus on the populated cells.Thus, biomolecular diffusion is restricted in the vicinity of the cells where collagen fibers are highly condensed.

View Article: PubMed Central - PubMed

Affiliation: Department of Life and Environment Engineering, Faculty of Environmental Engineering, The University of Kitakyushu, 1-1 Hibikino, Wakamatsu, Kitakyushu, Fukuoka, Japan ; Department of Mechanical Science and Bioengineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka, Japan.

ABSTRACT
The extracellular matrix (ECM) comprises the heterogeneous environment outside of cells in a biological system. The ECM is dynamically organized and regulated, and many biomolecules secreted from cells diffuse throughout the ECM, regulating a variety of cellular processes. Therefore, investigation of the diffusive behaviors of biomolecules in the extracellular environment is critical. In this study, we investigated the diffusion coefficients of biomolecules of various sizes, measuring from 1 to 10 nm in radius, by fluorescence correlation spectroscopy in contracted collagen gel caused by fibroblasts, a traditional culture model of dynamic rearrangement of collagen fibers. The diffusion coefficients of the biomolecules in control collagen gel without cells decreased slightly as compared to those in solution, while the diffusion coefficients of biomolecules in the contracted gel at the cell vicinity decreased dramatically. Additionally, the diffusion coefficients of biomolecules were inversely correlated with molecular radius. In collagen gels populated with fibroblasts, the diffusion coefficient at the cell vicinity clearly decreased in the first 24 h of culture. Furthermore, molecular diffusion was greatly restricted, with a central focus on the populated cells. By using the obtained diffusion coefficients of biomolecules, we calculated the collagen fiber condensation ratio by fibroblasts in the cell vicinity at 3 days of culture to represent a 52-fold concentration. Thus, biomolecular diffusion is restricted in the vicinity of the cells where collagen fibers are highly condensed.

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The contraction process of the cell-populated collagen gel.Gel contraction was evaluated by measuring gel weight. One milliliter of collagen solution including 2.5×105 TIG-1 cells was plated on a 35-mm culture dish and gelated. After gelation, the cell-populated collagen gel was cultured. The collagen gel weight was measured after removing culture medium from the dish. Each point is the mean ± standard deviation (n = 4). *p<0.01 vs. initial collagen gel weight.
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pone-0082382-g002: The contraction process of the cell-populated collagen gel.Gel contraction was evaluated by measuring gel weight. One milliliter of collagen solution including 2.5×105 TIG-1 cells was plated on a 35-mm culture dish and gelated. After gelation, the cell-populated collagen gel was cultured. The collagen gel weight was measured after removing culture medium from the dish. Each point is the mean ± standard deviation (n = 4). *p<0.01 vs. initial collagen gel weight.

Mentions: First, we evaluated the process of collagen gel contraction, which we used as a model for the dynamic steps of cell-dependent construction of the heterogeneous environment of the ECM. In this study, in order to allow for measurement of the contracted collagen gel by CLSM, we used collagen gels tightly attached to the surface of glass-based culture dishes or plates. Then, we evaluated the gel contraction process macroscopically by determining the weight of the collagen gel (Fig. 2). The initial collagen content in the gel was only 0.21% (w/v), and thus almost all of the gel contained culture media. Therefore, the gel contraction process could be simply evaluated by measuring the change in the weight of the collagen gel in the culture dish. The collagen gel weight decreased with culture time, and after 9 days in culture, the weight of the gel decreased to approximately 0.4-fold of its original weight (Fig. 2). Rapid contraction occurred during the first 24 h of culture, and this process was same as previous gel contraction study [32]. The collagen gel was macroscopically contracted by about 1.8-fold after 3 days of culture with TIG-1 cell.


Measurement of biomolecular diffusion in extracellular matrix condensed by fibroblasts using fluorescence correlation spectroscopy.

Kihara T, Ito J, Miyake J - PLoS ONE (2013)

The contraction process of the cell-populated collagen gel.Gel contraction was evaluated by measuring gel weight. One milliliter of collagen solution including 2.5×105 TIG-1 cells was plated on a 35-mm culture dish and gelated. After gelation, the cell-populated collagen gel was cultured. The collagen gel weight was measured after removing culture medium from the dish. Each point is the mean ± standard deviation (n = 4). *p<0.01 vs. initial collagen gel weight.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842966&req=5

pone-0082382-g002: The contraction process of the cell-populated collagen gel.Gel contraction was evaluated by measuring gel weight. One milliliter of collagen solution including 2.5×105 TIG-1 cells was plated on a 35-mm culture dish and gelated. After gelation, the cell-populated collagen gel was cultured. The collagen gel weight was measured after removing culture medium from the dish. Each point is the mean ± standard deviation (n = 4). *p<0.01 vs. initial collagen gel weight.
Mentions: First, we evaluated the process of collagen gel contraction, which we used as a model for the dynamic steps of cell-dependent construction of the heterogeneous environment of the ECM. In this study, in order to allow for measurement of the contracted collagen gel by CLSM, we used collagen gels tightly attached to the surface of glass-based culture dishes or plates. Then, we evaluated the gel contraction process macroscopically by determining the weight of the collagen gel (Fig. 2). The initial collagen content in the gel was only 0.21% (w/v), and thus almost all of the gel contained culture media. Therefore, the gel contraction process could be simply evaluated by measuring the change in the weight of the collagen gel in the culture dish. The collagen gel weight decreased with culture time, and after 9 days in culture, the weight of the gel decreased to approximately 0.4-fold of its original weight (Fig. 2). Rapid contraction occurred during the first 24 h of culture, and this process was same as previous gel contraction study [32]. The collagen gel was macroscopically contracted by about 1.8-fold after 3 days of culture with TIG-1 cell.

Bottom Line: Additionally, the diffusion coefficients of biomolecules were inversely correlated with molecular radius.Furthermore, molecular diffusion was greatly restricted, with a central focus on the populated cells.Thus, biomolecular diffusion is restricted in the vicinity of the cells where collagen fibers are highly condensed.

View Article: PubMed Central - PubMed

Affiliation: Department of Life and Environment Engineering, Faculty of Environmental Engineering, The University of Kitakyushu, 1-1 Hibikino, Wakamatsu, Kitakyushu, Fukuoka, Japan ; Department of Mechanical Science and Bioengineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka, Japan.

ABSTRACT
The extracellular matrix (ECM) comprises the heterogeneous environment outside of cells in a biological system. The ECM is dynamically organized and regulated, and many biomolecules secreted from cells diffuse throughout the ECM, regulating a variety of cellular processes. Therefore, investigation of the diffusive behaviors of biomolecules in the extracellular environment is critical. In this study, we investigated the diffusion coefficients of biomolecules of various sizes, measuring from 1 to 10 nm in radius, by fluorescence correlation spectroscopy in contracted collagen gel caused by fibroblasts, a traditional culture model of dynamic rearrangement of collagen fibers. The diffusion coefficients of the biomolecules in control collagen gel without cells decreased slightly as compared to those in solution, while the diffusion coefficients of biomolecules in the contracted gel at the cell vicinity decreased dramatically. Additionally, the diffusion coefficients of biomolecules were inversely correlated with molecular radius. In collagen gels populated with fibroblasts, the diffusion coefficient at the cell vicinity clearly decreased in the first 24 h of culture. Furthermore, molecular diffusion was greatly restricted, with a central focus on the populated cells. By using the obtained diffusion coefficients of biomolecules, we calculated the collagen fiber condensation ratio by fibroblasts in the cell vicinity at 3 days of culture to represent a 52-fold concentration. Thus, biomolecular diffusion is restricted in the vicinity of the cells where collagen fibers are highly condensed.

Show MeSH
Related in: MedlinePlus