Oxygen concentration is a key parameter in tissue culture. In addition, oxygen diffusion through biomaterial scaffolds plays an important role in maintaining healthy tissues in vitro, as oxygen supply becomes a limiting factor during the growth of highly metabolic tissues and large tissue masses. This is mainly a result of the lack of vascularization in tissues cultured in vitro and the low solubility of oxygen in the culture medium. In order to engineer tunable scaffold materials that can improve oxygen delivery and sustain healthier tissues, understanding of oxygen transport through the scaffolds is required. Moreover, tools that can provide information regarding the changes in oxygen concentration at the surface and through the volume of the scaffolds are also required.
Here, we present the development and characterization of fluorescent oxygen-sensing microparticles designed for measuring oxygen concentration in microenvironments existing within standard cell culture and transparent three-dimensional (3D) cell scaffolds. In addition, we present their application in conjunction with an automated and non-invasive sensor analysis method, based on confocal microscopy, to measure oxygen concentration profiles through the volume of a hydrogel scaffold. The synthesis of the microparticles is a two-step process that employs poly(dimethylsiloxane) to encapsulate silica gel particles bound with an oxygen-sensitive luminophore, tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) dichloride, as well as a reference or normalization fluorophore, Nile blue chloride, that is insensitive to oxygen. Microparticle size (5-40 ƒÝm) was selected for microscale-mapping of oxygen concentration to allow measurements local to individual cells. Additionally, the size of the microparticle allows for their suspension through the volume of a large range of hydrogel scaffolds.
The versatility of the described oxygen-sensing microparticles has allowed for studies where the effective diffusivity of oxygen through various hydrogel scaffold materials was estimated. In this application, a parallel-plate flow-chamber system was employed to emulate a plane sheet with variable surface concentration, where diffusion of oxygen occurs from the bulk of the moving fluid to the matrix of the hydrogel scaffold. Confocal microscopy was used to obtain maps of fluorescence intensity through the volume of the scaffolds. Subsequently, a Stern-Volmer calibration of the microparticles was employed to obtain the oxygen concentration profiles through the scaffold, which were later used to estimate the oxygen effective diffusivity.
The described fluorescent microparticles will enable characterization of oxygen diffusion through new scaffold materials and comparison of such properties to those of other existing scaffold biomaterials. Knowledge of these properties can lead to better design of scaffold materials than can improve oxygen delivery during culture. Future studies will focus achieving dynamic correlations of local oxygen concentration with individual cell response in cultured engineered tissues.