Optimization and Validation of FePro Cell Labeling Method

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 µg/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 µg/ml and Pro 0.75 to 3 µg/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 ∼30–35 pg-iron/cell at 24 h when labeled with 100 µg/ml of Fe and 3 µg/ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved ∼10 pg-iron/cell at 48 h when labeled with 100 µg/ml of Fe and 3 µg/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 µg/ml of Fe and 3 µg/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 µg/ml of Fe and 3 µg/ml of Pro is effective in labeling cells for cellular MRI.

Ferumoxides–protamine sulfate is more effective than ferucarbotran for cell labeling: implications for clinically applicable cell tracking using MRI

“…Various modifications of labeling protocols using ferumoxides have been reported in order to decrease the occurrence of extracellular iron deposits, like the use of heparin washes, additional incubation time or a different timing of complex formation (7,12,13,16). We did not include these adjustments in our main experiments, in order to better compare both techniques.…”

“…Full Paper approximately 100% have been reported regarding human bone marrow stromal cell (hBMSC) labeling, both methods are hampered by the occurrence of extracellular iron nanoparticle aggregates (5,7,(12)(13)(14)(15)(16).…”

Fluorescent magnetic iron oxide nanoparticles for cardiac precursor cell selection from stromal vascular fraction and optimization for magnetic resonance imaging

“…13 We have effectively assembled different elements (PEG and mAb) into a magnetic nanoparticle (SPION) without altering their inherent properties and engineered a SPION-based nanoprobe through an electrostatic interaction. This nanoprobe is capable of selectively separating progenitor cells from a heterogeneous population of cells and subsequently enables the cells to be tracked through noninvasive molecular imaging.…”

“…21 We evaluated the rate of clearance of iron from labeled cells with concomitant assessment of their viability in long-term culture. Quantitative analysis of the iron content of the labeled cells, using a standard protocol by Janic et al 13 showed a gradual and steady decrease in iron concentration over time, suggesting possible clearance of iron by endogenous pathways. …”

“…The cells were maintained in culture for as many as 10 days and viability/presence of nanoprobe within the cells was evaluated on day 2, 6, and 10. Intracellular iron content of labeled cells was also measured according to the well-established protocol of Janic et al 13 (Supplementary materials, S6). Enriched labeled cells were induced using differentiation medium which consisted of low DMEM (L-DMEM), supplemented with fetal bovine serum (FBS) (2.0%), bFGF (10 ng/mL), and 5-azacytidine (3.0 µM), for 48 hours.…”