In Vitro Assessment of Fluorine Nanoemulsion-Labeled Hyaluronan-Based Hydrogels for Precise Intrathecal Transplantation of Glial-Restricted Precursors.

Abstract

We studied the feasibility of labeling hydrogel scaffolds with a fluorine nanoemulsion for 19F- magnetic resonance imaging (MRI) to enable non-invasive visualization of their precise placement and potential degradation. Hyaluronan-based hydrogels (activated hyaluronan, HA) withincreasing concentrations of fluorine nanoemulsion (V-sense) were prepared to measure thegelation time and oscillatory stress at 1h and 7days after the beginning of gelation. All biomechanical measurements were conducted with an ARES 2 rheometer. Diffusion of fluorine from the hydrogel: Three hydrogels in various Vs to HA volumetric ratios (1:50, 1:10, and 1:5) were prepared in duplicate. Hydrogels were incubated at 37°C. To inducediffusion, three hydrogels were agitated at 1000rpm. 1H and 19F MRI scans were acquired at 1, 3, 7days and 2months after gel preparation on a Bruker Ascend 750 scanner. To quantify fluorine content, scans were analyzed usingVoxel Tracker 2.0. Assessment ofcell viability in vitro and in vivo: Luciferase-positive mouseglial-restricted progenitors (GRPs) were embedded in 0:1, 1:50, 1:10, and 1:5 Vs:HA mixtures (final cell concentration =1 × 107/ml). For the in vitro assay, mixtures were placedin 96-wells plate in triplicate and bioluminescence was measured after1, 3, 7, 14, 21, and 28days. For in vivo experiments, Vs/HA mixtures containing GRPs were injected subcutaneously in SCID mice and BLI was acquired at 1, 3, 7, and 14dayspost-injection. Mixing of V-sense at increasing ratios of 1:50, 1:10, and 1:5 v/v of fluorine/activated hyaluronan (HA) hydrogel gradually elongated the gelation time from 194s for non-fluorinated controls to 304s for 1:5 V-sense:HA hydrogels, while their elastic properties slightly decreased. There was no release of V-sense from hydrogels maintained in stationary conditions over 2months. The addition of V-sense positively affected in vitro survival of scaffolded GRPs in a dose-dependent manner. These results show that hydrogel fluorination does not impair its beneficial properties for scaffolded cells, which may be used to visualize scaffolded GRP transplants with 19F MRI.