Figure 1.
Schematic diagram of probe diffusion in the contracted collagen gel.
Collagen fibers are well condensed in the area surrounding the cell due to cellular activity. Biomolecules diffuse throughout the collagen fiber-containing space, in which the concentration of the collagen fibers varies from region to region. Diffusion behaviors of the biomolecules are probably affected by the local concentration of the collagen fibers. By measuring the diffusion behaviors of the biomolecules locally, we can develop a better understanding of the behaviors of these molecules in the heterogeneous ECM and of the physical environment surrounding the cells.
Figure 2.
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.
Figure 3.
Phase contrast micrographs of TIG-1 cells.
TIG-1 cells were cultured on regular tissue culture (TC) substrate or in collagen gel. The cell suspension in the culture medium or collagen solution was plated on a 35-mm culture dish. Each time is shown at the upper left of the corresponding micrograph. Bar: 100 µm.
Figure 4.
Fluorescent micrographs of the condensation process of collagen fibrils by populated fibroblasts.
TIG-1 cells were cultured in FITC-labeled collagen gels (0.7 mg/mL). The populated cells were seen as completely black objects (arrowheads). Each time is shown at the upper left of the corresponding micrograph. FITC-labeled collagen fibers were condensed into the area surrounding the cell. Bar: 50 µm.
Figure 5.
Diffusion coefficients of biomolecules in each condition.
The diffusion coefficients (D) of biomolecules (Alexa488 alkyne, Alexa488 dextran [3 kDa], FITC dextran [4 kDa], Alexa488 dextran [10 kDa], FITC dextran [40 kDa], Alexa488 streptavidin, and Alexa488 IgG) were measured by FCS in the solution, collagen gel (2.1 mg/mL), or cell populated and condensed collagen gel. In the condensed collagen gel, the D of the biomolecules was measured at the surface of cells (∼500 nm). Each D was plotted versus each molecular Stokes radius, r. Each point is the mean ± standard deviation (n = 10 or 20). The lines show the inverse correlation between D and r. * p<0.01 vs. D in the solution of the same biomolecule.
Table 1.
Diffusion coefficients and Stokes radii of the probes.
Figure 6.
Changes in biomolecular diffusion during the culture of cells in collagen gel.
The D of Alexa488 dextran (10 kDa) was measured at the surface of the cells (∼500 nm) on each culture day. The data for day 0 was that of cells incubated with the collagen gel for 30 min. Each point is the mean ± standard deviation (n = 20). * p<0.01 vs. D at day 0.
Figure 7.
Diffusion coefficients of biomolecules in the space surrounding the gel-containing cell surface.
The D of Alexa488 dextran (10 kDa) was measured at various distances from the cell surface of cells grown in the contracted gel at 3 days of culture. Each point is mean ± standard deviation (n = 10). * p<0.01 vs. D at the distance of 0.5 µm from the cell. # p<0.01 vs. D at the distance of 1.0 µm from the cell.
Figure 8.
Relative values of diffusion coefficients in collagen gel and in solution.
The relative values of diffusion coefficients in collagen gel (DG) compared to those in solution (D0) for each biomolecule are plotted versus the Stokes radius, r. Each D was already plotted in Fig. 5. The open circles show the relative data of the normal collagen gel (2.1 mg/mL; DGi/D0), and the closed diamonds show the relative data of the contracted collagen gel (DGc/D0). Each point is the mean ± standard deviation (n = 10 or 20).