Fluorescent Microparticles for Sensing Cell Microenvironment Oxygen Levels within 3D Scaffolds

Abstract

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. The microparticle synthesis employs poly(dimethylsiloxane) to encapsulate silica gel particles bound with an oxygen-sensitive luminophore as well as a reference or normalization fluorophore that is insensitive to oxygen. We developed a rapid, automated and non-invasive sensor analysis method based on fluorescence microscopy to measure oxygen concentration in a hydrogel scaffold. We demonstrate that the microparticles are non-cytotoxic and that their response is comparable to that of a traditional dissolved oxygen meter. Microparticle size (5-40 μm) was selected for microscale-mapping of oxygen concentration to allow measurements local to individual cells. Two methods of calibration were evaluated and revealed that the sensor system enables characterization of a range of hypoxic to hyperoxic conditions relevant to cell and tissue biology (i.e., pO 2 10-160 mm Hg). The calibration analysis also revealed that the microparticles have a high fraction of quenched luminophore (0.90 ± 0.02), indicating that the reported approach provides significant advantages for sensor performance. This study thus reports a versatile oxygen-sensing technology that enables future correlations of local oxygen concentration with individual cell response in cultured engineered tissues.

Use of Culture Geometry to Control Hypoxia-Induced Vascular Endothelial Growth Factor Secretion from Adipose-Derived Stem Cells: Optimizing a Cell-Based Approach to Drive Vascular Growth

“…As the master regulator of transcriptional responses to low oxygen conditions, HIF activation may be the most meaningful indicator of cellular hypoxia. 31 Tracking HIF activity rather than oxygen concentration/distribution, as in other techniques, [32][33][34] provides evidence of a behavioral response. This is useful, as the oxygen concentration required to trigger a behavioral effect can vary between cells or even within the same cell under different microenvironmental conditions.…”

Fluorescent oxygen sensitive microbead incorporation for measuring oxygen tension in cell aggregates

“…OSBs were synthesized according to a protocol, adapted from Acosta et al [26] and is composed of three steps (first two steps identical to Acosta et al). Beads (~1x10 7 ) were incubated at low temperature (4°C) in 1 ml maintenance medium (DMEM:F12, 5% FBS, 1% A/A, 3x10 -8 M sodium selenite, 10 µg/ml human transferrin) supplemented with 50 µl of an Alexa Fluor 488 labelled streptavidin solution (2 mg/ml) for ~24 hours.…”

“…The application of paramagnetic probes, such as trityl radicals, combined with electron paramagnetic resonance (EPR) is a very promising approach that provides reasonable resolution images (~10-30 µm) within acceptable acquisition times (minutes) [25]. Dual luminophore oxygen-sensing beads (OSBs) also offer great possibilities for measuring oxygen tension in a fast and non-interfering manner and have been developed for measuring local oxygen tension in (poly(ethylene glycol)dimethylacrylate) hydrogel carriers [26,27]. As the absence of controlled, site-specific delivery methods for these probes have prevented so far their practical application to cellular systems, we developed a non-invasive method for spatially controlled microbead incorporation into dense cell aggregate environments and assessed its feasibility to measure local oxygen tension.…”

Tracking Hypoxic Signaling in Encapsulated Stem Cells

“…43,44 The oxygen concentrations in the media and inside the scaffolds have been measured using oxygensensing dyes, oxygen microelectrodes, fluorescent probes, and microparticles. [45][46][47][48][49][50] Mathematical modeling approaches have been developed accounting for scaffold geometry and local oxygen tension to predict oxygen gradients inside the scaffolds. 51 These techniques to measure or predict oxygen concentration offer a great deal of information to the tissue engineering community.…”

“…Several techniques have been applied to measure the spatial distribution of oxygen within tissues using bloodgas analyzers, dissolved oxygen probes, and fluorescent/ luminescent particles. [48][49][50]60,61 These systems offer high levels of sensitivity and specificity, but do not show how the cells respond to their local oxygen environment. The marker system described here can complement such techniques and offers great potential as a tool to track biological responses in tissue engineering studies.…”