The purpose of this study was to develop an injectable hydrogel labeled with MRI nanosensor to facilitate the cell delivery to the brain with MRI monitoring of cell status. Previously, we showed that nanosensor-labeled hydrogels can be used to immunoprotect transplanted cells and sense cell viability [1-3]. Here, we customized the hydrogel focusing on reducing its stiffness, thus better supporting migration of glial restricted progenitor (GRP) cells following intracerebral delivery. Furthermore, we utilized CEST contrast at 5 ppm to improve specificity of the nanosensor. CEST-MRI protocol for phantoms: Alginate hydrogels labeled with liposomal nanosensors were imaged on a vertical bore 11.7T Bruker Avance system at 37°C. A modified rapid acquisition with relaxation enhancement (RARE) sequence including a saturation pulse was used to acquire saturation images at different irradiation frequency, which were used to generate the z-spectrum in each voxel. A slice thickness of 1 mm was used, and the typical imaging parameters were: TE=4.3ms, RARE factor=16, matrix size=128x64, NA=2, and the field of view was 13x13x1mm. Two sets of saturation images were acquired. First, we acquired frequency images to map the spatial distribution of B0. Water Saturation Shift Referencing (WASSR) mapping was employed [4, 5], for which we used a saturation pulse length of 500ms, saturation field strength (B1) of 0.5µT, and saturation frequency increment of 0.1ppm. Secondly, we acquired CEST images. The acquisition time per frequency point was 12s for frequency maps (TR=1.5s) and 48s for CEST images (TR=6s). For CEST data, we used a saturation pulse length of 4s, B1 values of 1.6,2.4, 3.6,4.7 and 5.9µT, and a frequency increment of 0.2ppm. Nanosensor-labeled hydrogel preparation: Alginate (Novamatrix) was dissolved in buffered saline, and then mixed with liposomes [6] containing either L-arginine (2ppm label) [1] or barbituric acid (5ppm label) [7]. GRP cells isolated from the spinal cord of E13.5 old PLP-GFP+/Luc+ mice were cultured in growth medium for 15-21days. CEST-MRI protocol in vivo: Rag2 mice (8w, 23–25g) were positioned in a custom made holder equipped with a heating system, anesthetized with isoflurane (1-1.5%) and MRI was performed on a horizontal bore 11.7T BIOSPEC system with a 23mm volume coil. The acquisition parameters for in vivo study: TR=5.0s, RARE factor=8, tsat=3s, B1=3.6 or 4.7μT, slice thickness=1mm, acquisition matrix size=128×64, FOV=16×16mm, NA=2. WASSR offset range =±2ppm (0.1ppm steps) and z-spectra offset range=±6ppm (0.2ppm steps). All data were processed using custom-written Matlab scripts. In vivo bioluminescence (BL) imaging: was performed for two groups of mice, one with (Gel, n=2) and one without (Control, n=1) the presence of hydrogel. All mice were imaged on days 0,1,5,8 and 14 after transplantation and 25min after luciferin injection (150mg/kg i.p.) using an IVIS 200 optical imaging system (Caliper Life Sciences) [1]. BL images were processed using Xenogen Living Imaging software. We optimized the stiffness and gelation time of alginate-based hydrogel, assuring there is sufficient time for injection and to favor the survival of the GRP cells. As shown in Fig.1, we imaged the cell viability of GRPs using conventional BL imaging after transplantation into Rag2 mice, BL signal is higher in the presence of hydrogel compared to the graft without hydrogel from day 5 onwards. The BL signal in normalized radiance was 62% higher than that of the control in two weeks (n=2), showing the hydrogel supports cell survival. Moreover, the CEST-MRI contrast of the nanosensor-labeled hydrogel is at 5ppm, which is away from the endogenous contrast of 1-4ppm in the brain, thus is favorable for neural applications. The Rag2 mouse transplanted with cells embedded in hydrogel labeled with nanosensors detectable at 5ppm showed strong contrast as compared to the surrounding brain tissue (contrast of the hydrogel to the brain (Gel:Brain=1.6, Fig. 2A)), while the hydrogel-labeled with nanosenors at 2ppm was not so conspicuous (Gel:Brain=1.2, Fig. 2B). The lower contrast to background ratio at 2ppm is probably due to the high endogenous CEST background and direct saturation and conventional magnetization transfer effects in the brain. We are now studying the contrast change over time after transplantation and examine if we can use this approach to longitudinally monitor engrafted cells.In this study, we have developed an injectable alginate-based hydrogel to facilitate the delivery and monitoring of cells grafted into the brain. This hydrogel formulation can be robustly labeled with liposomes as the nanosensors for imaging transplanted cells using CEST-MRI. The contrast is at 5ppm, which is removed from the endogenous CEST contrast at 1-4ppm, thus facilitating more specific detection. This favors application to cell replacement therapies into the brain.