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.

Previously, we reported that a nanoneedle of 200 nm diameter manipulated by an atomic force microscope apparatus could be inserted into a living cell. The insertion probabilities varied according to cell type. However, the nanoneedle was never successfully inserted into artificial liposomes. In the current study, we found that the stress fibers and actin filaments comprising the plasmalemmal undercoat are important, determining factors as to whether a nanoneedle can be successfully inserted into a cell. Depolymerization of microtubules increased both the number of stress fibers and insertion efficiency in NRK cells. These results indicate that the insertion efficiency of a nanoneedle (200 nm in diameter) into a cell with a smaller actin meshwork in its plasmalemmal undercoat is enhanced and the formation of stress fibers obviously contributes to this incremental enhancement. These facts are not only important as technical information to improve the efficiency of cell manipulation but also as observations of the mechanical properties of the native cell cortex.

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