(584k) “Shake-n-Incubate”: Examining the Effects of Orbital Shaking On Iron Oxide Nanoparticle Uptake for D1 Cell Labeling

Labeling stem cells with superparamagnetic iron oxide nanoparticles (SPIOs) enables clinicians to assess the bioavailability and localization of transplanted cells through MR imaging. To facilitate the cellular uptake of SPIOs for cell therapies, previous protocols optimized the concentration, size, surface charge, and targeting capability of SPIOs, often coupled with a transfection agent.  However, these approaches have limited degrees of success, in part due to concerns regarding transfection agent cytotoxicity. In this study, we sought to control cell labeling by manipulating the cellular microenvironment while keeping the intrinsic properties and concentration of the nanoparticles constant . To this end, we prepared SPIO clusters formed by the hydrophobic association between SPIOs and poly(2-hydroxyethyl aspartamide) substituted with hydrophobic octadecyl chains.  These water-stable, monodisperse SPIO clusters have a T₂ relaxivity comparable to Feridex, an FDA-approved MR contrast agent. We incubated bone marrow-derived mesenchymal stem cells in cell culture media suspended with SPIO clusters while shaking the cell culture well at varying rates.  The cells exposed to the orbital shaking at 50 rpm took up more SPIOs than cells cultured in static culture wells, as quantified through ICP-OES.  In addition, cells cultured under shaking at 50 rpm exhibited lower T₂ signal than those cultured without any external mechanical flow. Interestingly, the role of orbital shaking on SPIO uptake became minimal when serum concentration in the cell culture media was decreased.  This intriguing protocol will be extended to label a wide array of therapeutic cells, in turn improving transplanted cell tracking and, ultimately, the success of cell therapies.