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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 T and AC133+ hHSCs cells.Microphotography DAB-enhanced PB staining of non-labeled T-cells (A, bar = 50 µm) and T cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h (B, bar = 50 µm). Note the intracellular localization of FePro complexes that appear as the dark brown granules on electron microscopic images (arrows). Electron microscopy images of T-cells (C–D, bar = 1 µm) and AC133+ HSCs (F–G, bar = 1 µm) cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h. Prussian blue staining of AC133+ HSCs cells (E, bar = 20 µm).
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pone-0005873-g003: FePro labeled T and AC133+ hHSCs cells.Microphotography DAB-enhanced PB staining of non-labeled T-cells (A, bar = 50 µm) and T cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h (B, bar = 50 µm). Note the intracellular localization of FePro complexes that appear as the dark brown granules on electron microscopic images (arrows). Electron microscopy images of T-cells (C–D, bar = 1 µm) and AC133+ HSCs (F–G, bar = 1 µm) cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h. Prussian blue staining of AC133+ HSCs cells (E, bar = 20 µm).

Mentions: To assess the effectiveness and validate the short incubation time-low FePro concentration revised technique in suspension type cells, activated CD3+ T-cells and CD 133+ hHSC were incubated with 100 µg/ml of Fe and 3 µg/ml of Pro for 4 h. T-cell immunological stimulation, manifested as proliferative T-cell response, was induced by co-culturing T-cells with γ-irradiated (35–40 Gy) tumor primed dendritic cells. After 7 days of co-culture, T-cells were incubated with FePro and analyzed for labeling efficiency, intracellular iron concentration and cell viability. As expected labeling efficiency, assessed by DAB-enhanced PB staining (Figure 3A–B) was greater than 95% and electron microscopy analysis showed intracytoplasmic/endosomal localization of internalized iron complexes (Figure 3C–D). In addition, intracellular iron concentration was significantly (p<0.05) higher compared to non-labeled, control T-cells (4.3 pg/cell vs. 0.19 pg/cell). Trypan blue analysis showed that the 4 hr incubation time labeling with FePro did not have any significant effect on T-cell viability (53%), compared to non-labeled control cells (55%). Most importantly, FePro labeled T-cells exhibited significant proliferative response after secondary immunological stimulation induced by 5 days co-culture with γ-irradiated tumor primed dendritic cells. This response, analyzed by MTT assay and expressed as optical density, exhibited no difference between labeled (0.279±0.0015) and control, non-labeled T-cells (0.260±0.003). These data indicate that the 4 hour incubation time and lower concentration of FePro in media did not alter T-cell functional status.


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 T and AC133+ hHSCs cells.Microphotography DAB-enhanced PB staining of non-labeled T-cells (A, bar = 50 µm) and T cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h (B, bar = 50 µm). Note the intracellular localization of FePro complexes that appear as the dark brown granules on electron microscopic images (arrows). Electron microscopy images of T-cells (C–D, bar = 1 µm) and AC133+ HSCs (F–G, bar = 1 µm) cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h. Prussian blue staining of AC133+ HSCs cells (E, bar = 20 µm).
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Related In: Results  -  Collection

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pone-0005873-g003: FePro labeled T and AC133+ hHSCs cells.Microphotography DAB-enhanced PB staining of non-labeled T-cells (A, bar = 50 µm) and T cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h (B, bar = 50 µm). Note the intracellular localization of FePro complexes that appear as the dark brown granules on electron microscopic images (arrows). Electron microscopy images of T-cells (C–D, bar = 1 µm) and AC133+ HSCs (F–G, bar = 1 µm) cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h. Prussian blue staining of AC133+ HSCs cells (E, bar = 20 µm).
Mentions: To assess the effectiveness and validate the short incubation time-low FePro concentration revised technique in suspension type cells, activated CD3+ T-cells and CD 133+ hHSC were incubated with 100 µg/ml of Fe and 3 µg/ml of Pro for 4 h. T-cell immunological stimulation, manifested as proliferative T-cell response, was induced by co-culturing T-cells with γ-irradiated (35–40 Gy) tumor primed dendritic cells. After 7 days of co-culture, T-cells were incubated with FePro and analyzed for labeling efficiency, intracellular iron concentration and cell viability. As expected labeling efficiency, assessed by DAB-enhanced PB staining (Figure 3A–B) was greater than 95% and electron microscopy analysis showed intracytoplasmic/endosomal localization of internalized iron complexes (Figure 3C–D). In addition, intracellular iron concentration was significantly (p<0.05) higher compared to non-labeled, control T-cells (4.3 pg/cell vs. 0.19 pg/cell). Trypan blue analysis showed that the 4 hr incubation time labeling with FePro did not have any significant effect on T-cell viability (53%), compared to non-labeled control cells (55%). Most importantly, FePro labeled T-cells exhibited significant proliferative response after secondary immunological stimulation induced by 5 days co-culture with γ-irradiated tumor primed dendritic cells. This response, analyzed by MTT assay and expressed as optical density, exhibited no difference between labeled (0.279±0.0015) and control, non-labeled T-cells (0.260±0.003). These data indicate that the 4 hour incubation time and lower concentration of FePro in media did not alter T-cell functional status.

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