Method to Measure Local Diffusibility Distribution in Cultural Agarose Hydrogel Medium Using Fluorescent Nanoparticles

Hydrogel is an essential material in cell culture. The hydrogel network structures work as cell scaffolds. Cells attach to them and change the signaling inside. In addition, hydrogel water includes and supplies biological small molecules, such as nutrients and growth factors, as well as biological macromolecules, such as proteins and exosomes, to cells. The molecules regulate cellular behaviors. The diffusibility is important for the supply of the molecules and is affected by the space size. Hydrogel network structures have random pore sizes at the nanoscale; therefore, local diffusibility is necessary. Local diffusibilities in hydrogel have been investigated using fluorescent dyes at the molecular scale and using nanoparticles at the nanoscale. The local diffusibilities of nanoscale objects whose sizes are comparable to biological macromolecules in a cultural hydrogel medium should also be studied. Here, we proposed a method to measure local diffusibility by tracking nanoparticles suspended in a hydrogel, calculating the diffusion coefficients using their Brownian motions. To measure local diffusibilities, nanoparticles moving by free Brownian motions are necessary. The network structure of agarose was labeled with fluorescent dye, and the pore size was measured at around 400 nm in a 1.5% agarose hydrogel by using super-resolution fluorescent microscopy. We observed Brownian motions of 25 nm nanoparticles in the hydrogel and selected the nanoparticles with free Brownian motions by coefficient of determination. We could calculate local diffusion coefficients in the 1.5% agarose hydrogel and map local diffusibilities.