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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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The labeling efficiency is determined by the incubation time and iron concentrations.Prolonged incubation and high iron concentrations resulted in more labeling. In the present study, the optimized incubation condition for NPCs was 75 µg/ml over 48 hours. Higher concentrations than 75 µg/ml tended to cause undesired aggregation (see the 100 µg/ml panel).
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pone-0056125-g001: The labeling efficiency is determined by the incubation time and iron concentrations.Prolonged incubation and high iron concentrations resulted in more labeling. In the present study, the optimized incubation condition for NPCs was 75 µg/ml over 48 hours. Higher concentrations than 75 µg/ml tended to cause undesired aggregation (see the 100 µg/ml panel).

Mentions: The phenotype of the SVZ cells was identified by immunostaining. As shown in Fig. 1A, a majority of the cells within the spheres were positive for nestin, a commonly used marker for neural progenitors [23]. The immunohistochemical staining was performed as follows. P1 neurospheres were cytospun onto glass slides. After fixation in 4% paraformaldehyde for 1 hour at room temperature (RT)., the slides were incubated with mouse anti-rat nestin (1∶100; Stemcell technologies Inc., Vancouver, Canada) in PBS containing 0.1% Triton X and 10% normal goat serum overnight at 4°C, followed by incubation in rhodamine conjugated goat anti-mouse IgG (1∶200; Dako, Denmark) at RT for 1 hour. The cells were further stained with DAPI (100 ng/mL; Dako, Denmark). A fluorescent microscope was used to image the staining. Culture media and supplements were purchased from Gibco (Grang Island, USA). Chemicals were from Sigma-Aldrich (St. Louis, USA).


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)

The labeling efficiency is determined by the incubation time and iron concentrations.Prolonged incubation and high iron concentrations resulted in more labeling. In the present study, the optimized incubation condition for NPCs was 75 µg/ml over 48 hours. Higher concentrations than 75 µg/ml tended to cause undesired aggregation (see the 100 µg/ml panel).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056125-g001: The labeling efficiency is determined by the incubation time and iron concentrations.Prolonged incubation and high iron concentrations resulted in more labeling. In the present study, the optimized incubation condition for NPCs was 75 µg/ml over 48 hours. Higher concentrations than 75 µg/ml tended to cause undesired aggregation (see the 100 µg/ml panel).
Mentions: The phenotype of the SVZ cells was identified by immunostaining. As shown in Fig. 1A, a majority of the cells within the spheres were positive for nestin, a commonly used marker for neural progenitors [23]. The immunohistochemical staining was performed as follows. P1 neurospheres were cytospun onto glass slides. After fixation in 4% paraformaldehyde for 1 hour at room temperature (RT)., the slides were incubated with mouse anti-rat nestin (1∶100; Stemcell technologies Inc., Vancouver, Canada) in PBS containing 0.1% Triton X and 10% normal goat serum overnight at 4°C, followed by incubation in rhodamine conjugated goat anti-mouse IgG (1∶200; Dako, Denmark) at RT for 1 hour. The cells were further stained with DAPI (100 ng/mL; Dako, Denmark). A fluorescent microscope was used to image the staining. Culture media and supplements were purchased from Gibco (Grang Island, USA). Chemicals were from Sigma-Aldrich (St. Louis, USA).

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
Related in: MedlinePlus