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Optimization and validation of FePro cell labeling method.

Branislava Janic¹, Ali M Rad, Elaine K Jordan

¹Cellular and Molecular Imaging Laboratory, Department of Radiology, Henry Ford Hospital, Detroit, MI, USA. bjanic@rad.hfh.edu

|June 12, 2009

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A revised method for magnetic cell labeling using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) significantly reduces incubation time to 4 hours. This optimized technique enhances labeling efficiency and MRI signal intensity while maintaining cell viability.

Area of Science:

Biomedical Engineering
Cell Biology
Magnetic Resonance Imaging

Background:

Current ferumoxides (Fe)-protamine (Pro) sulfate (FePro) cell labeling requires long incubation (>12-16 hours) and high protamine doses.
This leads to large extracellular FePro complexes, difficult removal, and suboptimal T2* weighted MRI signal intensity.

Purpose of the Study:

To revise the FePro cell labeling method for shorter incubation times and improved MRI signal.
To investigate the use of lower protamine doses and direct addition of Fe and Pro to cells.

Main Methods:

Human glioma (U251) and leukemia (THP-1) cell lines were used for dose- and time-dependent labeling experiments.
Labeling efficiency was assessed via Prussian blue staining and intracellular iron concentration measurements.

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Cell viability and extracellular complex formation were evaluated after simple washes.

Main Results:

Over 95% of cells were labeled with the revised method.
Comparable labeling efficiency was achieved within 4 hours, significantly shorter than the previous >12-16 hours.
The new technique showed minimal extracellular FePro complexes and did not significantly affect cell viability.
Effective labeling was validated in human T-cells, hematopoietic stem cells, bone marrow stromal cells, and mouse neuronal stem cells.

Conclusions:

The revised FePro cell labeling technique is highly efficient, requiring only a 4-hour incubation period.
This optimized method improves labeling efficiency and MRI signal quality without compromising cell viability or function.
The direct addition of Fe and Pro to cells with reduced protamine concentration offers a superior alternative for cellular MRI applications.