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Accelerated stem cell labeling with ferucarbotran and protamine.

Golovko DM, Henning T, Bauer JS, Settles M, Frenzel T, Mayerhofer A, Rummeny EJ, Daldrup-Link HE - Eur Radiol (2009)

Bottom Line: Electron microscopy confirmed intracellular iron oxide uptake in lysosomes.Relaxation times correlated with results from ICP-AES.Our results show internalization of ferucarbotran can be accelerated in MSCs with protamine, an approved heparin antagonist and potentially clinically applicable uptake-enhancing agent.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology & Biomedical Imaging, University of California San Francisco, San Francisco, CA, USA.

ABSTRACT

Objective: To develop and characterize a clinically applicable, fast and efficient method for stem cell labeling with ferucarbotran and protamine for depiction with clinical MRI.

Methods: The hydrodynamic diameter, zeta potential and relaxivities of ferucarbotran and varying concentrations of protamine were measured. Once the optimized ratio was found, human mesenchymal stem cells (MSCs) were labeled at varying incubation times (1-24 h). Viability was assessed via Trypan blue exclusion testing. 150,000 labeled cells in Ficoll solution were imaged with T1-, T2- and T2*-weighted sequences at 3 T, and relaxation rates were calculated.

Results: Varying the concentrations of protamine allows for easy modification of the physicochemical properties. Simple incubation with ferucarbotran alone resulted in efficient labeling after 24 h of incubation while assisted labeling with protamine resulted in similar results after only 1 h. Cell viability remained unaffected. R2 and R2* relaxation rates were drastically increased. Electron microscopy confirmed intracellular iron oxide uptake in lysosomes. Relaxation times correlated with results from ICP-AES.

Conclusion: Our results show internalization of ferucarbotran can be accelerated in MSCs with protamine, an approved heparin antagonist and potentially clinically applicable uptake-enhancing agent.

Show MeSH
Relaxivities of complexes of ferucarbotran (50 mg iron per ml) related to the molar concentration of iron and increasing concentrations of protamine [in labeling media (DMEM + 25% FCS), 1.5 T, 25°C]
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Fig2: Relaxivities of complexes of ferucarbotran (50 mg iron per ml) related to the molar concentration of iron and increasing concentrations of protamine [in labeling media (DMEM + 25% FCS), 1.5 T, 25°C]

Mentions: With increasing protamine concentration, and as such, increasing complex size, the r1 and r2 relaxivities decrease, whereas the r2* relaxivity increases (Fig. 2). This is in accordance with findings with contrast agents of different sizes [25]. There seems to be a plateau effect with higher concentrations of protamine; however, because of instable complex formation seen with concentrations above 10 μg/ml, we were unable to confirm this. We believe that a concentration of 5 μg/ml is optimal for uptake in MSCs as this combines a complex size and electrical charge that are not too large, are still negative and have favorable relaxivity properties. At a concentration of 5 μg/ml, the complexes displayed the following relaxivities (in DMEM + 25% FCS, 25°C, 1.5 T): r1 2.50 ± 0.13 s−1mM−1, r2 20.16 ± 5.04 s−1mM−1 and r2* 574.00 ± 10.64 s−1mM−1.Fig. 2


Accelerated stem cell labeling with ferucarbotran and protamine.

Golovko DM, Henning T, Bauer JS, Settles M, Frenzel T, Mayerhofer A, Rummeny EJ, Daldrup-Link HE - Eur Radiol (2009)

Relaxivities of complexes of ferucarbotran (50 mg iron per ml) related to the molar concentration of iron and increasing concentrations of protamine [in labeling media (DMEM + 25% FCS), 1.5 T, 25°C]
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2822227&req=5

Fig2: Relaxivities of complexes of ferucarbotran (50 mg iron per ml) related to the molar concentration of iron and increasing concentrations of protamine [in labeling media (DMEM + 25% FCS), 1.5 T, 25°C]
Mentions: With increasing protamine concentration, and as such, increasing complex size, the r1 and r2 relaxivities decrease, whereas the r2* relaxivity increases (Fig. 2). This is in accordance with findings with contrast agents of different sizes [25]. There seems to be a plateau effect with higher concentrations of protamine; however, because of instable complex formation seen with concentrations above 10 μg/ml, we were unable to confirm this. We believe that a concentration of 5 μg/ml is optimal for uptake in MSCs as this combines a complex size and electrical charge that are not too large, are still negative and have favorable relaxivity properties. At a concentration of 5 μg/ml, the complexes displayed the following relaxivities (in DMEM + 25% FCS, 25°C, 1.5 T): r1 2.50 ± 0.13 s−1mM−1, r2 20.16 ± 5.04 s−1mM−1 and r2* 574.00 ± 10.64 s−1mM−1.Fig. 2

Bottom Line: Electron microscopy confirmed intracellular iron oxide uptake in lysosomes.Relaxation times correlated with results from ICP-AES.Our results show internalization of ferucarbotran can be accelerated in MSCs with protamine, an approved heparin antagonist and potentially clinically applicable uptake-enhancing agent.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology & Biomedical Imaging, University of California San Francisco, San Francisco, CA, USA.

ABSTRACT

Objective: To develop and characterize a clinically applicable, fast and efficient method for stem cell labeling with ferucarbotran and protamine for depiction with clinical MRI.

Methods: The hydrodynamic diameter, zeta potential and relaxivities of ferucarbotran and varying concentrations of protamine were measured. Once the optimized ratio was found, human mesenchymal stem cells (MSCs) were labeled at varying incubation times (1-24 h). Viability was assessed via Trypan blue exclusion testing. 150,000 labeled cells in Ficoll solution were imaged with T1-, T2- and T2*-weighted sequences at 3 T, and relaxation rates were calculated.

Results: Varying the concentrations of protamine allows for easy modification of the physicochemical properties. Simple incubation with ferucarbotran alone resulted in efficient labeling after 24 h of incubation while assisted labeling with protamine resulted in similar results after only 1 h. Cell viability remained unaffected. R2 and R2* relaxation rates were drastically increased. Electron microscopy confirmed intracellular iron oxide uptake in lysosomes. Relaxation times correlated with results from ICP-AES.

Conclusion: Our results show internalization of ferucarbotran can be accelerated in MSCs with protamine, an approved heparin antagonist and potentially clinically applicable uptake-enhancing agent.

Show MeSH