Trimodal imaging platform tracks implanted pancreatic islets
23 Oct 2018
Type I diabetes sufferers use insulin injections to control their blood sugar; but in unstable diabetes, blood sugar levels swing sharply and unpredictably, causing frequent episodes of hypo- or hyper-glycaemia. The transplantation of functioning pancreatic islet cells into the liver is a possible treatment for unstable diabetes, but current grafts are partially or fully rejected over time.
Artificial scaffolds show promising transplantation efficiency, but to better understand and therefore improve islet engraftment, more precise methods of non-invasively monitoring grafts are required. A number of imaging modalities have been used to track islet engraftments, each with its own limitations. Therefore, scientists are looking to combine imaging modalities to monitor islet distribution, number and viability.
Daniel Jirák and colleagues at the Institute for Clinical and Experimental Medicine, Charles University and Radboud University have previously combined MRI and fluorescent probes within nanoparticles. However, in this and other MRI studies, there is signal persistence in tissues thought to create false positives.
Therefore in this recent work, Jirák’s group has included a third imaging modality to confirm cell viability – bioluminescence. The aim is to resolve the MRI false-positive dilemma and provide a novel platform for in vivo insights into islet engraftment and rejection (Mol. Imaging Biol. 10.1007/s11307-018-1270-3).
The bioluminescence trick
Live cells genetically altered to express luciferase emit a bioluminescent signal in the presence of oxygen and adenosine triphosphate. These are crucial properties of live cells, making bioluminescence a marker of cell viability. To leverage bioluminescence in pancreatic islet implants, Jirák’s group bred luciferase transgenic Lewis rats, and then removed the bioluminescent pancreatic islet cells for further modification.
The researchers prepared bimodal poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles by single-emulsion solvent evaporation, incorporating liquid perfluorocarbon marker for F-19 MR imaging, and indocyanine green dye for near-infrared fluorescent imaging. Nanoparticles were endocytosed into islet donor cells, which they tested in vitro.
Staining of intact membrane proteins showed that nanoparticle labelling had not affected islet cell viability, and a glucose stimulated insulin secretion test proved functionality. The researchers also tested the three imaging modalities in vitro, using an optical imager for fluorescent and bioluminescent readings and a 4.7T MR scanner with a homemade radiofrequency coil for MRI. The team established that good signal-to-noise ratios could be achieved for long exposure MRI, and that fluorescence imaging was sensitive in much shorter time frames.
Proof is in the in vivo study
Jirák’s team then transplanted prepared islet cells into an artificial scaffold that had been subcutaneously added into the abdomen of male adult Lewis rats. The three imaging modalities were performed at multiple time-points throughout a two-week period. The team found that the bioluminescent and F-19 MRI signals correlated well, showing islet cell presence in scaffolds throughout, with maximum signal on day four.
This correlation suggests that no false-positive signal occurred, contradicting other studies where F-19 probe persisted after cell death. Jirák and colleagues think that this apparent contradiction could be explained by increased washout of fluorine probe from dead islet cells, perhaps caused by differences in labelling formulation of PLGA nanoparticles, and the greater vascularization experienced by islets in the artificial mesh. Histological examination supports this theory, as macrophages, important in clearance, were found within the meshes.
Fluorescence, the third imaging modality, showed the strongest signal on day one but then rapidly decreased over the first week. This signal quenching suggests instability of the fluorescent dye, perhaps due to thermal degradation or leakage. The group performed long-term incubations in vitro and showed a decrease in fluorescent signal over time, confirming the dye’s instability and limited applicability for longitudinal studies.
Complementary modalities
Jirák’s group proved that these three imaging modalities complement one another in tracking pancreatic islet cell implants. The fluorescent dye is highly sensitive, quickly confirming implantation success; this is compared with the low sensitivity of F-19 MRI that requires one-hour image acquisition times. Despite this, F-19 MRI has been shown to be sensitive enough to quantify islet cell numbers and thereby estimate graft size. The need for genetic engineering to establish bioluminescence in islet cells limits this modality to non-human experiments for the moment, but in this context it will be useful in determining cell viability.
The team now hopes that this novel platform for in vivo multimodal tracking will provide kinetic insights into the process of engraftment and rejection of islet cell transplants in future studies.
Louisa Cockbill is a science writer based in the UK
23/10/2018 from physicsworld.com
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