Pre-Microporation Improves Outcome of Pancreatic Islet Labelling for Optical and 19F MR Imaging

Abstract

BackgroundIn vitro labelling of cells and small cell structures is a necessary step before in vivo monitoring of grafts. We modified and optimised a procedure for pancreatic islet labelling using bimodal positively charged poly(lactic-co-glycolic acid) nanoparticles with encapsulated perfluoro crown ethers and indocyanine green dye via microporation and compared the method with passive endocytosis.ResultsPancreatic islets were microporated using two pulses at various voltages. We tested a standard procedure (poration in the presence of nanoparticles) and a modified protocol (pre-microporation in a buffer only, and subsequent islet incubation with nanoparticles on ice for 10 min).We compared islet labelling by microporation with labelling by endocytosis, i.e. pancreatic islets were incubated for 24 h in a medium with suspended nanoparticles.In order to verify the efficiency of the labelling procedures, we used 19F magnetic resonance imaging, optical fluorescence imaging and confocal microscopy.The experiment confirmed that microporation, albeit fast and effective, is invasive and may cause substantial harm to islets. To achieve sufficient poration and to minimise the reduction of viability, the electric field should be set at 20 kV/m (two pulses, 20 ms each).Poration in the presence of nanoparticles was found to be unsuitable for the nanoparticles used. The water suspension of nanoparticles (which served as a surfactant) was slightly foamy and microbubbles in the suspension were responsible for sparks causing the destruction of islets during poration. However, pre-microporation (poration of islets in a buffer only) followed by 10-min incubation with nanoparticles was safer.ConclusionsFor labelling of pancreatic islets using poly(lactic-co-glycolic acid) nanoparticles, the modified microporation procedure with low voltage was found to be safer than the standard microporation procedure. The modified procedure was fast, however, efficiency was lower compared to endocytosis.

A Trimodal Imaging Platform for Tracking Viable Transplanted Pancreatic Islets In Vivo: F-19 MR, Fluorescence, and Bioluminescence Imaging

“…Labeling by endocytosis maintained the viability and functionality of the labeled islets, which was also proved by a comparable bioluminescence signal between labeled and unlabeled islets. Sufficient labeling of islets via endocytosis using PLGA-NPs was also confirmed by confocal microscopy in our previous paper [ 29 ]. Since the bioluminescence signal is dependent on the presence of oxygen and adenosine triphosphate, only viable cells emit photons in a bioluminescence reaction [ 28 ].…”

“…Although fluorescence imaging was found to be a more sensitive method for cell tracking compared to F-19 MRI, we observed a steep decline in the in vivo fluorescence signal. Quenching of the fluorescence signal originating from islets labeled by the high concentration of PLGA-NPs has been described previously [ 29 ], but the fluorescence signal used in this model decreased rapidly within 4 days after transplantation. Fluorescence of labeled islets decreased to a noise level within 1 week, while F-19 MRI and bioluminescence signals decreased only partially.…”

“…Since the bioluminescence signal is dependent on the presence of oxygen and adenosine triphosphate, only viable cells emit photons in a bioluminescence reaction [ 28 ]. Published studies on the following topics attest to the high viability and insulin secretion of labeled islets: non-toxicity of probes based on perfluorocarbons [ 33 ] and the safety of selected labeling methods [ 29 ]. The same nanoparticles have been used to label various subsets of primary human dendritic cells in preparation for clinical application [ 34 ].…”

“…The F-19 MRI signal is influenced not only by F-19 concentration, but also by the distance from the coil. Other errors in quantification of the F-19 MRI signal may arise from filtering during post-processing, low measurement matrices, and Fourier transform, potentially resulting in partial signal dispersion within the whole measurement matrix [ 29 , 41 ]. To overcome these sensitivity problems and to improve F-19 SNR, various data acquisition methods, such as compressed sensing [ 39 ] and ultrashort echo sequences [ 42 ], have been proposed, which could reduce imaging time while maintaining or even improving the SNR.…”

“…In our previous study, we focused on optimizing a protocol for PI labeling using PLGA-based nanoparticles (PLGA-NPs) containing both PFCE and the NIR probe indocyanine green (ICG). PIs were visualized in vitro using F-19 MRI and fluorescence imaging [ 29 ]. The aim of the current study was to monitor the fate of PIs labeled with bimodal PLGA-NPs after transplantation into artificial scaffolds using trimodal imaging (F-19 MRI, fluorescence, and bioluminescence imaging).…”