Optimization of a nanoparticle uptake protocol applied to amniotic-derived cells: unlocking the therapeutic potential.

Stem cell-based therapy implementation relies heavily on advancements in cell tracking. The present research has been designed to develop a gold nanorod (AuNR) labeling protocol applied to amniotic epithelial cells (AECs) leveraging the pro-regenerative properties of this placental stem cell source which is widely used for both human and veterinary biomedical regenerative applications, although not yet exploited with tracking technologies. Ovine AECs, in native or induced mesenchymal (mAECs) phenotypes via epithelial-mesenchymal transition (EMT), served as the model. Initially, various uptake methods validated on other sources of mesenchymal stromal cells (MSCs) were assessed on mAECs before optimization for AECs. Furthermore, the protocol was implemented by adopting the biological strategy of MitoCeption to improve endocytosis. The results indicate that the most efficient, affordable, and easy protocol leading to internalization of AuNRs in living mAECs recognized the combination of the one-step uptake condition (cell in suspension), centrifugation-mediated internalization method (G-force) and MitoCeption (mitochondrial isolated from mAECs). This protocol produced labeled vital mAECs within minutes, suitable for preclinical and clinical trials. The optimized protocol has the potential to yield feasible labeled amniotic-derived cells for biomedical purposes: up to 10 million starting from a single amniotic membrane. Similar and even higher efficiency was found when the protocol was applied to ovine and human AECs, thereby demonstrating the transferability of the method to cells of different phenotypes and species-specificity, hence validating its great potential for the development of improved biomedical applications in cell-based therapy and diagnostic imaging.