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Simple SPION incubation as an efficient intracellular labeling method for tracking neural progenitor cells using MRI.

Chen CC, Ku MC, D M J, Lai JS, Hueng DY, Chang C - PLoS ONE (2013)

Bottom Line: However, compelling evidence also shows that simple SPION incubation is not invariably ineffective.The labeling efficiency can be improved by prolonged incubation and elevated iron doses.The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Sciences, Academic Sinica, Taipei, Taiwan.

ABSTRACT
Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs.

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Intracellular labeling efficiency of simple SPION incubation.(A) Unlabeled NPCs stained with PB staining. (B) SPION-labeled NPCs. (C) Unlabeled NPCs under electronic microscopy. (D) SPION-labeled NPCs. Several endosomes or lysosomes loaded with SPION-like particles were observed in the cytoplasm, indicated by arrows. N indicates the nucleus. (E) Quantification of the loaded endosomes/lysosomes indicates the intracellular labeling efficiency of the simple incubation method.
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pone-0056125-g003: Intracellular labeling efficiency of simple SPION incubation.(A) Unlabeled NPCs stained with PB staining. (B) SPION-labeled NPCs. (C) Unlabeled NPCs under electronic microscopy. (D) SPION-labeled NPCs. Several endosomes or lysosomes loaded with SPION-like particles were observed in the cytoplasm, indicated by arrows. N indicates the nucleus. (E) Quantification of the loaded endosomes/lysosomes indicates the intracellular labeling efficiency of the simple incubation method.

Mentions: PB staining was absent in unlabeled NPCs (Fig. 3A) but abundant in SPION labeled NPCs (Fig. 3B). When examining the PB staining at 100X, the counterstain dye (nuclear fast red) was found not only in the nuclei as pink but also in the cytoplasm as light pink. The cytoplasmic light pink color helped to define the cell-cell boundaries, and thus confirmed the intracellular localization of SPIONs. Electron microscopy reveals no appearance of nanoparticles in the cytoplasm of the unlabeled NPCs (Fig. 3C). By contrast, in the labeled NPCs, several vesicles loaded with particles likely to be SPIONs were observed (Fig. 3D). The vesicles resembled endosomes or lysosomes. Quantification of the loaded vesicles in the unlabeled and labeled NPCs is shown in Fig. 3E. Unlabeled cells have no SPION-loaded endo/lysosomes. Only unloaded endo/lysosomes were observed.


Simple SPION incubation as an efficient intracellular labeling method for tracking neural progenitor cells using MRI.

Chen CC, Ku MC, D M J, Lai JS, Hueng DY, Chang C - PLoS ONE (2013)

Intracellular labeling efficiency of simple SPION incubation.(A) Unlabeled NPCs stained with PB staining. (B) SPION-labeled NPCs. (C) Unlabeled NPCs under electronic microscopy. (D) SPION-labeled NPCs. Several endosomes or lysosomes loaded with SPION-like particles were observed in the cytoplasm, indicated by arrows. N indicates the nucleus. (E) Quantification of the loaded endosomes/lysosomes indicates the intracellular labeling efficiency of the simple incubation method.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585319&req=5

pone-0056125-g003: Intracellular labeling efficiency of simple SPION incubation.(A) Unlabeled NPCs stained with PB staining. (B) SPION-labeled NPCs. (C) Unlabeled NPCs under electronic microscopy. (D) SPION-labeled NPCs. Several endosomes or lysosomes loaded with SPION-like particles were observed in the cytoplasm, indicated by arrows. N indicates the nucleus. (E) Quantification of the loaded endosomes/lysosomes indicates the intracellular labeling efficiency of the simple incubation method.
Mentions: PB staining was absent in unlabeled NPCs (Fig. 3A) but abundant in SPION labeled NPCs (Fig. 3B). When examining the PB staining at 100X, the counterstain dye (nuclear fast red) was found not only in the nuclei as pink but also in the cytoplasm as light pink. The cytoplasmic light pink color helped to define the cell-cell boundaries, and thus confirmed the intracellular localization of SPIONs. Electron microscopy reveals no appearance of nanoparticles in the cytoplasm of the unlabeled NPCs (Fig. 3C). By contrast, in the labeled NPCs, several vesicles loaded with particles likely to be SPIONs were observed (Fig. 3D). The vesicles resembled endosomes or lysosomes. Quantification of the loaded vesicles in the unlabeled and labeled NPCs is shown in Fig. 3E. Unlabeled cells have no SPION-loaded endo/lysosomes. Only unloaded endo/lysosomes were observed.

Bottom Line: However, compelling evidence also shows that simple SPION incubation is not invariably ineffective.The labeling efficiency can be improved by prolonged incubation and elevated iron doses.The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Sciences, Academic Sinica, Taipei, Taiwan.

ABSTRACT
Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs.

Show MeSH