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Optimization and validation of FePro cell labeling method.

Janic B, Rad AM, Jordan EK, Iskander AS, Ali MM, Varma NR, Frank JA, Arbab AS - PLoS ONE (2009)

Bottom Line: FePro labeling did not significantly affect cell viability.Labeling for 4 hours using 100 microg/ml of Fe and 3 microg/ml of Pro resulted in very efficient labeling of these cells, without impairing their viability and functional capability.The new technique with short incubation time using 100 microg/ml of Fe and 3 microg/ml of Pro is effective in labeling cells for cellular MRI.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Imaging Laboratory, Department of Radiology, Henry Ford Hospital, Detroit, MI, USA. bjanic@rad.hfh.edu

ABSTRACT
Current method to magnetically label cells using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) is based on generating FePro complexes in a serum free media that are then incubated overnight with cells for the efficient labeling. However, this labeling technique requires long (>12-16 hours) incubation time and uses relatively high dose of Pro (5-6 microg/ml) that makes large extracellular FePro complexes. These complexes can be difficult to clean with simple cell washes and may create low signal intensity on T2* weighted MRI that is not desirable. The purpose of this study was to revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. Human tumor glioma (U251) and human monocytic leukemia cell (THP-1) lines were used as model systems for attached and suspension cell types, respectively and dose dependent (Fe 25 to 100 microg/ml and Pro 0.75 to 3 microg/ml) and time dependent (2 to 48 h) labeling experiments were performed. Labeling efficiency and cell viability of these cells were assessed. Prussian blue staining revealed that more than 95% of cells were labeled. Intracellular iron concentration in U251 cells reached approximately 30-35 pg-iron/cell at 24 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved approximately 10 pg-iron/cell at 48 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. Again, comparable labeling was observed after 4 h for the described FePro concentrations. FePro labeling did not significantly affect cell viability. There was almost no extracellular FePro complexes observed after simple cell washes. To validate and to determine the effectiveness of the revised technique, human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) and mouse neuronal stem cells (mNSC C17.2) were labeled. Labeling for 4 hours using 100 microg/ml of Fe and 3 microg/ml of Pro resulted in very efficient labeling of these cells, without impairing their viability and functional capability. The new technique with short incubation time using 100 microg/ml of Fe and 3 microg/ml of Pro is effective in labeling cells for cellular MRI.

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FePro labeled THP-1 and U-251 cells.Microphotography of cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h and stained using PB method to visualize the intracellular iron incorporation in THP-1 cells (A, bar = 50 µm) and U-251 cells (B, bar = 50 µm). Electron microscopy images of U251 cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h (C, D, bar = 1 µm) and control unlabeled U251 cells (E, bar = 1 µm). Note; extracellular FePro granules attached to the cell membranes and on their way to be engulfed and endocytosed (arrows) (C) and endocytosed intracellular iron within the endosomal compartment (arrows in circles) (D). Very little to no extracellular particles of FePro complexes seen compared to our previous method of labeling cells (F, G). Prussian blue staining of magnetically labeled hMSCs using our previous method showing extracellular complexes (F, bar = 10 µm) which are also confirmed by electron microscopic images (G bar = 2 µm). Extracellular complexes pointed by arrows in F–G.
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pone-0005873-g001: FePro labeled THP-1 and U-251 cells.Microphotography of cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h and stained using PB method to visualize the intracellular iron incorporation in THP-1 cells (A, bar = 50 µm) and U-251 cells (B, bar = 50 µm). Electron microscopy images of U251 cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h (C, D, bar = 1 µm) and control unlabeled U251 cells (E, bar = 1 µm). Note; extracellular FePro granules attached to the cell membranes and on their way to be engulfed and endocytosed (arrows) (C) and endocytosed intracellular iron within the endosomal compartment (arrows in circles) (D). Very little to no extracellular particles of FePro complexes seen compared to our previous method of labeling cells (F, G). Prussian blue staining of magnetically labeled hMSCs using our previous method showing extracellular complexes (F, bar = 10 µm) which are also confirmed by electron microscopic images (G bar = 2 µm). Extracellular complexes pointed by arrows in F–G.

Mentions: Qualitative analysis of FePro cellular uptake was performed on microscopic slide preparations of labeled cells that were stained with Prussian blue (PB). Representative bright light photomicrographs of THP-1 and U251 cells labeled with 100 µg/ml of Fe and 3 µg/ml of Pro for 4 h are shown in Figure 1A and 1B, respectively. Labeled cells exhibited abundant, heterogeneous uptake of FePro complexes that appeared as blue granules inside the cytoplasm on PB stained slides. Labeling efficiency analysis revealed that more than 95% of the cells were positive for PB. Electron microscopy (EM) analysis of U-251 cells labeled with 100 µg/ml of Fe and 3 µg/ml of Pro for 4 h, demonstrated the presence of fine FePro granules attached to the cell membranes and on their way to be macropinocytosed by the cells (Figure 1C and 1D), as well as intracellularly incorporated SPION within endosomes (Figure 1D). These data confirmed that 4 h time point was sufficient for generating intra-cytoplasmic, endosomal, iron oxide nanoparticle deposits (Figure 1B–D). In addition, EM demonstrated a decrease in the amount of large FePro complexes that accumulated extracellularly when we used our previously published labeling technique [6], [14] (Figure 1F–G). After labeling with 100 µg/ml of Fe and 3 µg/ml of Pro, intracellular iron concentration in U251 cells reached the maximum of ∼35 pg-iron/cell at 24 h time point. The highest iron concentrations in U-251 cells labeled with 50 µg/ml of Fe and 1.5 µg/ml of Pro and 25 µg/ml of Fe and 0.75 µg/ml of Pro were observed between 4–24 h after labeling (Figure 2A). After labeling U251 cells for 4 hrs with 100 µg/ml of Fe and 3 µg/ml of Pro, these cells exhibited significantly higher intracellular Fe concentrations compared to non-labeled control cells or cells incubated with 25–50 µg/ml of Fe and 0.75–1.5 µg/ml of Pro (p<0.05). In THP-1 cells the highest intracellular Fe concentrations were observed at 48 h in cells incubated with 100 µg/ml of Fe and 3 µg/ml of Pro or with 25 µg/ml of Fe and 0.75 µg/ml of Pro. THP-1 cells incubated with 50 µg/ml of Fe and 1.5 µg/ml of Pro exhibited peak intracellular Fe levels at 24 h time point. However, incubating THP-1 cells for 4 h with 100 µg/ml of Fe and 3 µg/ml of Pro resulted in significantly higher intracellular Fe concentrations (p<0.05) compared to two other FePro concentrations used (Figure 2B).


Optimization and validation of FePro cell labeling method.

Janic B, Rad AM, Jordan EK, Iskander AS, Ali MM, Varma NR, Frank JA, Arbab AS - PLoS ONE (2009)

FePro labeled THP-1 and U-251 cells.Microphotography of cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h and stained using PB method to visualize the intracellular iron incorporation in THP-1 cells (A, bar = 50 µm) and U-251 cells (B, bar = 50 µm). Electron microscopy images of U251 cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h (C, D, bar = 1 µm) and control unlabeled U251 cells (E, bar = 1 µm). Note; extracellular FePro granules attached to the cell membranes and on their way to be engulfed and endocytosed (arrows) (C) and endocytosed intracellular iron within the endosomal compartment (arrows in circles) (D). Very little to no extracellular particles of FePro complexes seen compared to our previous method of labeling cells (F, G). Prussian blue staining of magnetically labeled hMSCs using our previous method showing extracellular complexes (F, bar = 10 µm) which are also confirmed by electron microscopic images (G bar = 2 µm). Extracellular complexes pointed by arrows in F–G.
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Related In: Results  -  Collection

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pone-0005873-g001: FePro labeled THP-1 and U-251 cells.Microphotography of cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h and stained using PB method to visualize the intracellular iron incorporation in THP-1 cells (A, bar = 50 µm) and U-251 cells (B, bar = 50 µm). Electron microscopy images of U251 cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h (C, D, bar = 1 µm) and control unlabeled U251 cells (E, bar = 1 µm). Note; extracellular FePro granules attached to the cell membranes and on their way to be engulfed and endocytosed (arrows) (C) and endocytosed intracellular iron within the endosomal compartment (arrows in circles) (D). Very little to no extracellular particles of FePro complexes seen compared to our previous method of labeling cells (F, G). Prussian blue staining of magnetically labeled hMSCs using our previous method showing extracellular complexes (F, bar = 10 µm) which are also confirmed by electron microscopic images (G bar = 2 µm). Extracellular complexes pointed by arrows in F–G.
Mentions: Qualitative analysis of FePro cellular uptake was performed on microscopic slide preparations of labeled cells that were stained with Prussian blue (PB). Representative bright light photomicrographs of THP-1 and U251 cells labeled with 100 µg/ml of Fe and 3 µg/ml of Pro for 4 h are shown in Figure 1A and 1B, respectively. Labeled cells exhibited abundant, heterogeneous uptake of FePro complexes that appeared as blue granules inside the cytoplasm on PB stained slides. Labeling efficiency analysis revealed that more than 95% of the cells were positive for PB. Electron microscopy (EM) analysis of U-251 cells labeled with 100 µg/ml of Fe and 3 µg/ml of Pro for 4 h, demonstrated the presence of fine FePro granules attached to the cell membranes and on their way to be macropinocytosed by the cells (Figure 1C and 1D), as well as intracellularly incorporated SPION within endosomes (Figure 1D). These data confirmed that 4 h time point was sufficient for generating intra-cytoplasmic, endosomal, iron oxide nanoparticle deposits (Figure 1B–D). In addition, EM demonstrated a decrease in the amount of large FePro complexes that accumulated extracellularly when we used our previously published labeling technique [6], [14] (Figure 1F–G). After labeling with 100 µg/ml of Fe and 3 µg/ml of Pro, intracellular iron concentration in U251 cells reached the maximum of ∼35 pg-iron/cell at 24 h time point. The highest iron concentrations in U-251 cells labeled with 50 µg/ml of Fe and 1.5 µg/ml of Pro and 25 µg/ml of Fe and 0.75 µg/ml of Pro were observed between 4–24 h after labeling (Figure 2A). After labeling U251 cells for 4 hrs with 100 µg/ml of Fe and 3 µg/ml of Pro, these cells exhibited significantly higher intracellular Fe concentrations compared to non-labeled control cells or cells incubated with 25–50 µg/ml of Fe and 0.75–1.5 µg/ml of Pro (p<0.05). In THP-1 cells the highest intracellular Fe concentrations were observed at 48 h in cells incubated with 100 µg/ml of Fe and 3 µg/ml of Pro or with 25 µg/ml of Fe and 0.75 µg/ml of Pro. THP-1 cells incubated with 50 µg/ml of Fe and 1.5 µg/ml of Pro exhibited peak intracellular Fe levels at 24 h time point. However, incubating THP-1 cells for 4 h with 100 µg/ml of Fe and 3 µg/ml of Pro resulted in significantly higher intracellular Fe concentrations (p<0.05) compared to two other FePro concentrations used (Figure 2B).

Bottom Line: FePro labeling did not significantly affect cell viability.Labeling for 4 hours using 100 microg/ml of Fe and 3 microg/ml of Pro resulted in very efficient labeling of these cells, without impairing their viability and functional capability.The new technique with short incubation time using 100 microg/ml of Fe and 3 microg/ml of Pro is effective in labeling cells for cellular MRI.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Imaging Laboratory, Department of Radiology, Henry Ford Hospital, Detroit, MI, USA. bjanic@rad.hfh.edu

ABSTRACT
Current method to magnetically label cells using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) is based on generating FePro complexes in a serum free media that are then incubated overnight with cells for the efficient labeling. However, this labeling technique requires long (>12-16 hours) incubation time and uses relatively high dose of Pro (5-6 microg/ml) that makes large extracellular FePro complexes. These complexes can be difficult to clean with simple cell washes and may create low signal intensity on T2* weighted MRI that is not desirable. The purpose of this study was to revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. Human tumor glioma (U251) and human monocytic leukemia cell (THP-1) lines were used as model systems for attached and suspension cell types, respectively and dose dependent (Fe 25 to 100 microg/ml and Pro 0.75 to 3 microg/ml) and time dependent (2 to 48 h) labeling experiments were performed. Labeling efficiency and cell viability of these cells were assessed. Prussian blue staining revealed that more than 95% of cells were labeled. Intracellular iron concentration in U251 cells reached approximately 30-35 pg-iron/cell at 24 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved approximately 10 pg-iron/cell at 48 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. Again, comparable labeling was observed after 4 h for the described FePro concentrations. FePro labeling did not significantly affect cell viability. There was almost no extracellular FePro complexes observed after simple cell washes. To validate and to determine the effectiveness of the revised technique, human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) and mouse neuronal stem cells (mNSC C17.2) were labeled. Labeling for 4 hours using 100 microg/ml of Fe and 3 microg/ml of Pro resulted in very efficient labeling of these cells, without impairing their viability and functional capability. The new technique with short incubation time using 100 microg/ml of Fe and 3 microg/ml of Pro is effective in labeling cells for cellular MRI.

Show MeSH
Related in: MedlinePlus