Cell Labeling Efficiency Research Articles

A cell penetrating peptide-modified magnetic/fluorescent probe for in vivo tracking of mesenchymal stem cells.

Difficulty in precise tracking of the in vivo distribution and migration of transplanted stem cells in a noninvasive way is a great hurdle in regenerative medicine research. Paramagnetic element-doped upconversion nanoparticles (UCNPs) emerge as a kind of promising dual-modal imaging agents owing to the possibility in combining strong contrast of MR imaging and high efficiency of fluorescence imaging. Herein a kind of Mn-doped NaYF4-based UCNPs (NaYF4: 20%Yb, 2%Er, 30%Mn) was developed and then hydrophilized by various coatings, including cell penetrating peptide, DNA, and SiO2 . The UCNPs were denoted as Pep/UCNPs, DNA/UCNPs, and SiO2 /UCNPs, respectively. Their potential in cell labeling and in vivo tracking applications was comprehensively explored. The results show that the Mn-doped UCNPs possess high biocompatibility, high cell labeling efficiency, high MR resolution, and importantly a single emission at 660 nm, within optical window for in vivo imaging of biological targets. Particularly, mesenchymal stem cells (MSCs) pre-labeled by cell penetrating peptide-modified UCNPs (Pep/UCNPs) can be precisely monitored in terms of their distribution in mice over a long period of time by simultaneous MR and fluorescent imaging, which provided a noninvasive and double-checking tool for investigating the destination of stem cells in tissue regeneration.

Biodistribution of 89Zr-oxine-labeled human bone marrow-derived mesenchymal stem cells by micro-PET/computed tomography imaging in Sprague-Dawley rats.

PurposeTo develop a method for labeling human bone marrow mesenchymal stem cells (hMSCs) with 89Zr-oxine to characterize the biodistribution characteristics of hMSCs in normal Sprague–Dawley (SD) rats in real-time by micro-PET–computed tomography (micro-PET/CT) imaging.Methods89Zr-oxine complex was synthesized from 89Zr-oxalate and 8-hydroxyquinoline (oxine). After hMSCs were labeled with the 89Zr-oxine complex, the radioactivity retention, viability, proliferation, apoptosis, differentiation, morphology, and phenotype of labeled cells were assessed. The biodistribution of 89Zr-oxine-labeled hMSCs in SD rats was tracked in real-time by micro-PET/CT imaging.ResultsThe cell labeling efficiency was 52.6 ± 0.01%, and 89Zr-oxine was stably retained in cells (66.7 ± 0.9% retention on 7 days after labeling). Compared with the unlabeled hMSCs, 89Zr-oxine labeling did not affect the biological characteristics of cells. Following intravenous administration in SD rats, labeled hMSCs mainly accumulated in the liver (7.35 ± 1.41% ID/g 10 days after labeling, n = 6) and spleen (8.48 ± 1.20% ID/g 10 days after labeling, n = 6), whereas intravenously injected 89Zr-oxalate mainly accumulated in the bone (4.47 ± 0.35% ID/g 10 days after labeling, n = 3).Conclusion89Zr-oxine labeling and micro-PET/CT imaging provide a useful and non-invasive method of assessing the biodistribution of cell therapy products in SD rats. The platform provides a foundation for us to further understand the mechanism of action and migration dynamics of cell therapy products.

Continuous Flow Labeling and In-Line Magnetic Separation of Cells

There is an identified need for point-of-care diagnostic systems for detecting and counting specific rare types of circulating cells in blood. By adequately labeling such cells with immunomagnetic beads and quantum dots, they can be efficiently collected magnetically for quantification using fluorescence methods. Automation of this process requires adequate mixing of the labeling materials with blood samples. A static mixing device can be employed to improve cell labeling efficiency and eliminate error-prone laboratory operations. Computational fluid dynamics (CFD) were utilized to simulate the flow of a labeling-materials/blood mixture through a 20-stage in-line static mixer of the interfacial-surface-generator type. Optimal fluid mixing conditions were identified and tested in a magnetic bead/tumor cell model, and it was found that labeled cells could be produced at 1.0 mL/min flow rate and fed directly into an in-line magnetic trap. The trap design consists of a dual flow channel with three bends and a permanent magnet positioned at the outer curve of each bend. The capture of labeled cells in the device was simulated using CFD, finite-element analysis and magnetophoretic mobility distributions of labeled cells. Testing with cultured CRL14777 human melanoma cells labeled with anti-CD146 1.5 μm diameter beads indicated that 90 ± 10% are captured at the first stage, and these cells can be captured when present in whole blood. Both in-line devices were demonstrated to function separately and together as predicted.

Study on cancer stem cell labeling and inhibition efficiency of LV3 nanocomplex in vitro

Study on cancer stem cell labeling and inhibition efficiency of LV3 nanocomplex in vitro

Enhanced and long-term CT imaging tracking of transplanted stem cells labeled with temperature-responsive gold nanoparticles.

Gold nanoparticles (AuNPs) have been extensively employed for computed tomography (CT) imaging in cell labeling and tracking because of their strong X-ray attenuation coefficient and excellent biocompatibility. However, the design and synthesis of stimuli-responsive AuNPs to modulate their endocytosis and exocytosis for optimal cell labeling and tracking are promising but challenging. Herein, we report an innovative labeling strategy based on temperature-responsive AuNPs (TRAuNPs) with high cell labeling efficiency and extended intracellular retention duration. We have manifested that the TRAuNP labeling imposes a negligible adverse effect on the function of human mesenchymal stem cells (hMSCs). Further experiment with idiopathic pulmonary fibrosis (IPF) model mice has demonstrated the feasibility of TRAuNP labeling for long time CT imaging tracking of transplanted hMSCs. What's more, the survival of transplanted hMSCs could also be monitored simultaneously using bioluminescence imaging after the expression of luciferase reporter genes. Therefore, we believe that this dual-modal labeling and tracking strategy enables visualization of the transplanted hMSCs in vivo, which may provide an important insight into the role of stem cells in the IPF therapy.

Multifunctional Dendrimer-Entrapped Gold Nanoparticles for Labeling and Tracking T Cells Via Dual-Modal Computed Tomography and Fluorescence Imaging

Nanosystems for monitoring and tracking T cells provide an important basis for evaluating the functionality and efficacy of T cell-based immunotherapy. To this end, we designed herein an efficient nanoprobe for T cell monitoring and tracking using poly(amidoamine) (PAMAM) dendrimer-entrapped gold nanoparticles (Au DENPs) conjugated with Fluo-4 for dual-mode computed tomography (CT) and fluorescence imaging. In this study, PAMAM dendrimers of generation 5 (G5) were modified with hydroxyl-terminated polyethylene glycol (PEG) and then used to entrap 2.0 nm Au NPs followed by acetylation of the excess amine groups on the dendrimer surface. Subsequently, the calcium ion probe was covalently attached to the dendrimer nanohybrids through the PEG hydroxyl end groups to gain the functional {(Au0)25-G5.NHAc-(PEG)14-(Fluo-4)2} nanoprobe. This nanoprobe had excellent water solubility, high X-ray attenuation coefficient, and good cytocompatibility in the given concentration range, as well as a high T cell labeling efficiency. Confocal microscopy and flow cytometry results demonstrated that the nanoprobe was able to fluorescently sense activated T cells. Moreover, the nanoprobe was able to realize both CT and fluorescence imaging of subcutaneously injected T cells in vivo. Thus, the developed novel dendrimer-based nanosystem may hold great promise for advancing and improving the clinical application of T cell-based immunotherapy.

Dual-Modal Imaging-Guided Precise Tracking of Bioorthogonally Labeled Mesenchymal Stem Cells in Mouse Brain Stroke.

Noninvasive and precise stem cell tracking after transplantation in living subject is very important to monitor both stem cell destinations and their in vivo fate, which is closely related to their therapeutic efficacy. Herein, we developed bicyclo[6.1.0]nonyne (BCN)-conjugated glycol chitosan nanoparticles (BCN-NPs) as a delivery system of dual-modal stem cell imaging probes. Near-infrared fluorescent (NIRF) dye Cy5.5 was chemically conjugated to the BCN-NPs, and then oleic acid-coated superparamagnetic iron oxide nanoparticles (OA-Fe3O4 NPs) were encapsulated into BCN-NPs, resulting in Cy5.5-labeled and OA-Fe3O4 NP-encapsulated BCN-NPs (BCN-dual-NPs). For bioorthogonal labeling of human adipose-derived mesenchymal stem cells (hMSCs), first, hMSCs were treated with tetra-acetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz) for generating azide (-N3) groups onto their surface via metabolic glycoengineering. Second, azide groups on the cell surface were successfully chemically labeled with BCN-dual-NPs via bioorthogonal click chemistry in vitro. This bioorthogonal labeling of hMSCs could greatly increase the cell labeling efficiency, safety, and imaging sensitivity, compared to only nanoparticle-derived labeling technology. The dual-modal imaging-guided precise tracking of bioorthogonally labeled hMSCs was tested in the photothrombotic stroke mouse model via intraparenchymal injection. Finally, BCN-dual-NPs-labeled hMSCs could be effectively tracked by their migration from the implanted site to the brain stroke lesion using NIRF/T2-weighted magnetic resonance (MR) dual-modal imaging for 14 days. Our observation would provide a potential application of bioorthogonally labeled stem cell imaging in regenerative medicine by providing safety and high labeling efficiency in vitro and in vivo.

Radiolabeling of cell membrane-based nano-vesicles with 14C-linoleic acid for robust and sensitive quantification of their biodistribution

The rapid development of biomimetic cell membrane-based nanoparticles is still overshadowed by many practical challenges, one of which is the difficulty to precisely measure the biodistribution of such nanoparticles. Currently, this challenge is mostly addressed using fluorescent techniques with limited sensitivity, or radioactive labeling methods, which rarely account for the nanoparticles themselves, but their payloads instead. Here we report the development of a robust method for the innate radioactive labeling of cells and membrane-based nanoparticles and their consequent sensitive detection and biodistribution measurements. The preclinical potential of this method was demonstrated with Nano-Ghosts (NGs), manufactured from the cytoplasmic membranes of mesenchymal stem cells cultured with radioactively-labeled linoleic acid and achieving a cell labeling efficiency of 36%. Radiolabeling did not affect the physiochemical properties of the NGs, which stably retained their radiolabels. Using radioactivity measurements, we are now able to determine precisely the amount of NGs uptaken by tissues and cells, thereby providing further support to our presumed active NG targeting mechanisms. Biodistribution studies comparing radiolabeled NGs to fluorescently-labeled ones have validated our method and revealed new information, which could not be obtained otherwise, regarding the NGs' unique kinetics and rapid clearance, supporting their excellent safety profiles. The reported approach may be expanded to other membrane-based entities to facilitate and hasten their preclinical development and be used in parallel with other labeling methods to provide different and additional information.

Ratiometric Barcoding for Mass Cytometry

Barcoding is of importance for high-throughput cellular and molecular analysis. A ratiometric barcoding strategy using lanthanide-coordinated polymer dots (Ln-Pdots) was developed for mass cytometric analysis. By using 3 metal isotopes and 4 ratio intensity levels, 16 barcodes were generated to code, and later decode, cell samples in mass cytometry. The ratiometric Ln-Pdot barcodes not only provided high-mass-signal intensities but also eliminated the bias caused by different concentrations of the labeling reagents/barcodes and run-to-run differences in cell labeling efficiency. The ability to distinguish clearly the 16 sets of labeled MCF-7 cells with mass cytometry demonstrated the excellent resolving power of the ratiometric Ln-Pdot barcodes. Furthermore, the results from barcoding PBMC samples via CD45-specific cellular targeting indicated that the ratiometric Ln-Pdot barcodes could facilitate mass cytometry in high-throughput and multiplexed analysis, especially with precious human samples.

Single-Step Metal-Free Grafting of Cationic Polymer Brushes on Fluorescent Nanodiamonds.

Cationic polymers are often employed in conjugation with nanomaterials, and the resultant hybrids are useful for various bioapplications. Here, a single-step metal-free method for the synthesis of fluorescent nanodiamonds (FNDs) conjugated with cationic polymer brushes is reported. Distinct from the common methods such as atom transfer radical polymerization and reversible addition fragmentation chain transfer, our ring-opening-polymerization-based method is simple and less time consuming and hazardous. Infrared spectroscopy, thermogravimetric analysis, zeta potential, and dynamic light scattering confirmed the synthesis. The produced FND-polymer brushes showed markedly higher cell labeling and internalization efficiency without noticeable cytotoxicity. Our method is general and applicable to other nanoparticles as well for uses in diverse research areas.

Improved sensitivity of cellular MRI using phase-cycled balanced SSFP of ferumoxytol nanocomplex-labeled macrophages at ultrahigh field.

PurposeThe purpose of this study was to investigate the feasibility and sensitivity of cellular magnetic resonance imaging (MRI) with ferumoxytol nanocomplex-labeled macrophages at ultrahigh magnetic field of 7 T.Materials and methodsTHP-1-induced macrophages were labeled using self-assembling heparin + protamine + ferumoxytol nanocomplexes which were injected into a gelatin phantom visible on both microscope and MRI. Susceptibility-weighted imaging (SWI) and balanced steady-state free precession (bSSFP) pulse sequences were applied at 3 and 7 T. The average, maximum intensity projection, and root mean square combined images were generated for phase-cycled bSSFP images. The signal-to-noise ratio and contrast-to-noise ratio (CNR) efficiencies were calculated. Ex vivo experiments were then performed using a formalin-fixed pig brain injected witĥ100 and ~1,000 labeled cells, respectively, at both 3 and 7 T.ResultsA high cell labeling efficiency (.90%) was achieved with heparin + protamine + ferumoxytol nanocomplexes. Less than 100 cells were detectable in the gelatin phantom at both 3 and 7 T. The 7 T data showed more than double CNR efficiency compared to the corresponding sequences at 3 T. The CNR efficiencies of phase-cycled bSSFP images were higher compared to those of SWI, and the root mean square combined bSSFP showed the highest CNR efficiency with minimal banding. Following co-registration of microscope and MR images, more cells (51/63) were detected by bSSFP at 7 T than at 3 T (36/63). On pig brain, botĥ100 and ~1,000 cells were detected at 3 and 7 T. While the cell size appeared larger due to blooming effects on SWI, bSSFP allowed better contrast to precisely identify the location of the cells with higher signal-to-noise ratio efficiency.ConclusionThe proposed cellular MRI with ferumoxytol nanocomplex-labeled macrophages at 7 T has a high sensitivity to detect, 100 cells. The proposed method has great translational potential and may have broad clinical applications that involve cell types with a primary phagocytic phenotype.

A promising magnetic resonance stem cell tracer based on natural biomaterials in a biological system: manganese(II) chelated to melanin nanoparticles.

BackgroundMelanin and manganese are both indispensable natural substances that play crucial roles in the human body. Melanin has been used as a multimodality imaging nanoplatform for biology science research because of its natural binding ability with metal ions (eg, 64Cu2+, Fe3+, and Gd3+). Because of its effects on T1 signal enhancement, Mn-based nanoparticles have been used in magnetic resonance (MR) quantitative cell tracking in vivo. Stem cell tracking in vivo is an essential technology used to characterize engrafted stem cells, including cellular viability, biodistribution, differentiation capacity, and long-term fate.MethodsIn the present study, manganese(II) ions chelated to melanin nanoparticles [MNP-Mn(II)] were synthesized. The characteristics, stem cell labeling efficiency, and cytotoxicity of the nanoparticles were evaluated. MR imaging of the labeled stem cells in vivo and in vitro were also further performed. In T1 relaxivity (r1), MNP-Mn(II) were significantly more abundant than Omniscan. Bone marrow-derived stem cells (BMSCs) can be labeled easily by coincubating with MNP-Mn(II), suggesting that MNP-Mn(II) had high biocompatibility.ResultsCell Counting Kit-8 assays revealed that MNP-Mn(II) had almost no cytotoxicity when used to label BMSCs, even with a very high concentration (1,600 µg/mL). BMSCs labeled with MNP-Mn(II) could generate a hyperintense T1 signal both in vitro and in vivo, and the hyperintense T1 signal in vivo persisted for at least 28 days.ConclusionTaken together, our results showed that MNP-Mn(II) possessed many excellent properties for potential quantitative stem cell tracking in vivo.

The use of pulsed magnetic fields to increase the uptake of iron oxide nanoparticles by living cells

Remote control of the interaction of magnetic nanoparticles with cells is fundamental to any potential downstream applications of magnetic nanoparticles such as gene and drug delivery vehicles and magnetic cell labeling. Thus, approaches based on the application of external magnetic fields to increase the efficiency of magnetic cell labeling are desirable. Here, we report a simple approach that enhances magnetic cell labeling using pulsed magnetic fields. The rate of uptake of superparamagnetic iron oxide nanoparticles (SPIONs) and transport across the cell membrane were enhanced upon application of a high intensity (7 T) short pulse width (∼15 μs) magnetic field. We present a quantitative analysis and mechanistic explanation of how a pulsed magnetic field influences the uptake of SPIONs by cells. Our findings offer insights into the mechanics of how pulsed magnetic fields can be effectively used to optimize magnetic cell labeling, which can provide a basis for better controlled biomedical applications of SPIONs.

Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector

Cloning of multiple genes in a single vector has greatly facilitated both basic and translational studies that require co-expression of multiple factors or multi-units of complex protein. Many strategies have been adopted, among which 2A “self-cleaving” peptides have garnered increased interest for their polycistronic nature, small size and high “cleavage” efficiency. However, broad application of 2 A peptides is limited by the lack of systematic comparison of different 2As alone or in combination. Here we characterized the effect of varying gene position and 2As on the expression of proteins encoded in bi-, tri-, or quad-cistronic constructs. Using direct cardiac reprogramming as an example, we further determined the effect of varied 2As on the efficiency of fluorescent cell labeling and cell fate conversion. We found that the expression of fluorophores decreased as it was moved towards the end of the construct while reprogramming was most efficient with the fluorophore at the second position. Moreover, quad-cistronic TPE2A constructs resulted in more efficient reprogramming than 3P2A or PTE2A constructs. We expect that the bi-, tri-, and quad-cistronic vectors constructed here and our results on protein expression ratios from different 2A constructs could serve to guide future utilization of 2A peptides in basic research and clinical applications.

Effective tracking of bone mesenchymal stem cells in vivo by magnetic resonance imaging using melanin-based gadolinium3+ nanoparticles.

Tracking transplanted stem cells is necessary to clarify cellular properties and improve transplantation success. In this study, we designed and synthesized melanin-based gadolinium3+ (Gd3+ )-chelate nanoparticles (MNP-Gd3+ ) of ∼7 nm for stem cell tracking in vivo. MNP-Gd3+ possesses many beneficial properties, such as its high stability and sensitivity, shorter T1 relaxation time, higher cell labeling efficiency, and lower cytotoxicity compared with commercial imaging agents. We found that the T1 relaxivity (r1 ) of MNP-Gd3+ was significantly higher than that of Gd-DTPA; the nanoparticles were taken up by bone mesenchymal stem cells (BMSCs) via endocytosis and were broadly distributed in the cytoplasm. Based on an in vitro MTT assay, no cytotoxicity of labeled stem cells was observed for MNP-Gd3+ concentrations of less than 800 µg/mL. Furthermore, we tracked MNP-Gd3+ -labeled BMSCs in vivo using 3.0T MRI equipment. After intramuscular injection, MNP-Gd3+ -labeled BMSCs were detected, even after four weeks, by 3T MRI. We concluded that MNP-Gd3+ nanoparticles at appropriate concentrations can be used to effectively monitor and track BMSCs in vivo. MNP-Gd3+ nanoparticles have potential as a new positive MRI contrast agent in clinical applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 131-137, 2017.

Labeling and long-term tracking of bone marrow mesenchymal stem cells in vitro using NaYF4:Yb3+,Er3+ upconversion nanoparticles

Mesenchymal stem cells (MSCs) hold great promise as cell therapy candidate in clinics. However, the underlying mechanisms remain elusive due to the lack of effective cell tracking approaches during therapeutic processes. In this study, we successfully synthesized and utilized NaYF4:Yb3+,Er3+ upconversion nanoparticles (UCNPs) to label and track rabbit bone marrow mesenchymal stem cells (rBMSCs) during the osteogenic differentiation in vitro. To improve their biocompatibility and cellular uptake, we modified the UCNPs with negatively-charged poly(acrylic acid) and positively-charged poly(allylamine hydrochloride) in turns (i.e., PAH-PAA-UCNPs). The effect of cellular uptake of UCNPs on the osteogenic differentiation of rBMSCs was systematically evaluated, and no significant difference was found between rBMSCs labeled with UCNPs (concentration range of 0–50μg/mL) and UCNPs-free rBMSCs in terms of cell viability, ALP activity, osteogenic protein expressions and production of mineralized nodules. Moreover, the PAH-PAA-UCNPs at a concentration of 50μg/mL exhibited the highest biocompatibility and stability, which could well track rBMSCs during the osteogenesis process. These results would provide a positive reference for the application of these lanthanide-doped UCNPs as fluorescent nanoprobes for stem cell tracking to further understand the mechanism of stem cell fate in tissue engineering and stem cell therapy. Statement of SignificanceUpconversion nanoparticles (UCNPs) have attracted increasing attention as alternative probes for tracking various types of cells including stem cells. The reported fluorapatite-based UCNPs with the needle-like morphology showed a little poor performance on stem cell tracking, which was possibly attributed to the low upconversion efficiency and cell labeling efficiency potentially due to nanomaterial composition, crystal structure and shape. Here, we synthesized the positively-charged NaYF4:Yb3+,Er3+ UCNPs with hexagonal phase and sphere-like morphology to enhance their upconversion efficiency, biocompatibility and cellular uptake, leading to a successful tracking of rBMSCs in osteogenesis process without impairing cell viability and differentiation capacity. This study provided a necessary reference for the application of UCNPs in stem cell tracking to better understand the mechanism of stem cell fate in tissue engineering, stem cell therapy, etc.

Improved biocompatibility and efficient labeling of neural stem cells with poly(L-lysine)-coated maghemite nanoparticles.

Background: Cell tracking is a powerful tool to understand cellular migration, dynamics, homing and function of stem cell transplants. Nanoparticles represent possible stem cell tracers, but they differ in cellular uptake and side effects. Their properties can be modified by coating with different biocompatible polymers. To test if a coating polymer, poly(L-lysine), can improve the biocompatibility of nanoparticles applied to neural stem cells, poly(L-lysine)-coated maghemite nanoparticles were prepared and characterized. We evaluated their cellular uptake, the mechanism of internalization, cytotoxicity, viability and proliferation of neural stem cells, and compared them to the commercially available dextran-coated nanomag®-D-spio nanoparticles.Results: Light microscopy of Prussian blue staining revealed a concentration-dependent intracellular uptake of iron oxide in neural stem cells. The methyl thiazolyl tetrazolium assay and the calcein acetoxymethyl ester/propidium iodide assay demonstrated that poly(L-lysine)-coated maghemite nanoparticles scored better than nanomag®-D-spio in cell labeling efficiency, viability and proliferation of neural stem cells. Cytochalasine D blocked the cellular uptake of nanoparticles indicating an actin-dependent process, such as macropinocytosis, to be the internalization mechanism for both nanoparticle types. Finally, immunocytochemistry analysis of neural stem cells after treatment with poly(L-lysine)-coated maghemite and nanomag®-D-spio nanoparticles showed that they preserve their identity as neural stem cells and their potential to differentiate into all three major neural cell types (neurons, astrocytes and oligodendrocytes).Conclusion: Improved biocompatibility and efficient cell labeling makes poly(L-lysine)-coated maghemite nanoparticles appropriate candidates for future neural stem cell in vivo tracking studies.

Magnetic resonance imaging of ultrasmall superparamagnetic iron oxide-labeled exosomes from stem cells: a new method to obtain labeled exosomes.

PurposeRecent findings indicate that the beneficial effects of adipose stem cells (ASCs), reported in several neurodegenerative experimental models, could be due to their paracrine activity mediated by the release of exosomes. The aim of this study was the development and validation of an innovative exosome-labeling protocol that allows to visualize them with magnetic resonance imaging (MRI).Materials and methodsAt first, ASCs were labeled using ultrasmall superparamagnetic iron oxide nanoparticles (USPIO, 4–6 nm), and optimal parameters to label ASCs in terms of cell viability, labeling efficiency, iron content, and magnetic resonance (MR) image contrast were investigated. Exosomes were then isolated from labeled ASCs using a standard isolation protocol. The efficiency of exosome labeling was assessed by acquiring MR images in vitro and in vivo as well as by determining their iron content. Transmission electron microscopy images and histological analysis were performed to validate the results obtained.ResultsBy using optimized experimental parameters for ASC labeling (200 µg Fe/mL of USPIO and 72 hours of incubation), it was possible to label 100% of the cells, while their viability remained comparable to unlabeled cells; the detection limit of MR images was of 102 and 2.5×103 ASCs in vitro and in vivo, respectively. Exosomes isolated from previously labeled ASCs retain nanoparticles, as demonstrated by transmission electron microscopy images. The detection limit by MRI was 3 µg and 5 µg of exosomes in vitro and in vivo, respectively.ConclusionWe report a new approach for labeling of exosomes by USPIO that allows detection by MRI while preserving their morphology and physiological characteristics.

Tracking and Quantification of Magnetically Labeled Stem Cells using Magnetic Resonance Imaging.

Stem cell based therapies have critical impacts on treatments and cures of diseases such as neurodegenerative or cardiovascular disease. In vivo tracking of stem cells labeled with magnetic contrast agents is of particular interest and importance as it allows for monitoring of the cells' bio-distribution, viability, and physiological responses. Herein, recent advances are introduced in tracking and quantification of super-paramagnetic iron oxide (SPIO) nanoparticles-labeled cells with magnetic resonance imaging, a noninvasive approach that can longitudinally monitor transplanted cells. This is followed by recent translational research on human stem cells that are dual-labeled with green fluorescence protein (GFP) and SPIO nanoparticles, then transplanted and tracked in a chicken embryo model. Cell labeling efficiency, viability, and cell differentiation are also presented.

Simple Methods for Tracking Stem Cells with (64)Cu-Labeled DOTA-hexadecyl-benzoate.

The purpose of this study was to evaluate (64)Cu-labeled hexadecyl-1,4,7,10-tetraazacyclododecane-tetraacetic acid-benzoate ((64)Cu-DOTA-HB) (1) as positron emission tomography (PET) radiotracer for stem cell imaging. Hexadecyl-DOTA-benzoate (DOTA-HB) (2) was efficiently labeled with (64)Cu (>99%), and cell labeling efficiency with adipose-derived stem cells (ADSCs) was over 50%. Labeling with 1 did not compromise cell viability. In the PET imaging, intramuscularly transplanted 1-labeled ADSCs were monitored for 18 h in normal rat heart. These results indicate that 1 can be utilized as a promising radiotracer for monitoring of transplanted stem cells.