Nuclei were labeled by 4,6-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich, 1.1000). least a month, indicating stable incorporation, a necessity for long-term imaging. Using a clinical 3T MRI, 1 103 haNSCs were visualized upon injection in a gel phantom, but detection limit was much lower (5 104 cells) in layer phantoms and using an imaging protocol feasible in a clinical scenario. Transcriptional analysis and fluorescence immunocytochemistry did not reveal a detrimental impact of VSOP labeling on important parameters of cellular physiology with cellular viability, stemness and neuronal differentiation potential remaining unaffected. This represents Lobetyolin a pivotal prerequisite with respect to clinical application of this method. MRI with different modalities including one being close to a potential clinical application. Results Security of Cell Labeling With VSOP To assess security of haNSC preparation, cryopreservation, and labeling (0.5 mM), or to detect any donor-dependent differences, cell viability was tested in the first step. No significant donor-dependent differences in cell viability between cells which underwent the labeling process with 0.5 mM (85C89%) and non-labeled control cells (91%, sample pooled from all patients) could be detected 1 day after labeling (Figure 1A). All samples could be included in onward experiments according to preset viability criteria ( 80%). Next, cell viability of haNSCs and Rabbit Polyclonal to RAD17 mESCs was compared 8 and 48 h after labeling with 0.5 and 1.5 mM VSOP, respectively (Determine 1B). Again, no significant differences in haNSCs viability between non-labeled control cells (95%), as well as 8 (87.5%) and 48 h (94.5%) after labeling became apparent. Viability of mESCs decreased slightly to 89% at 8 h and to 93.5% at 48 h after labeling. No viability differences were observed between 0.5 and 1.5 mM VSOP concentration. Open in a separate window Lobetyolin Physique 1 Cell viability of magnetically labeled haNSCs and mESCs (= 3 with 3 technical replicates each). (A) Trypan blue exclusion test showed no significant differences in viability of three different patient samples (labeling with 0.5 mM). (B) Trypan blue exclusion test 8 and 48 h after labeling showed no decrease in cell viability due to the labeling process. Efficacy of Magnetic Cell Labeling Incubation of haNSCs with 0.5 mM VSOP alone (simple) and additional lipofection resulted in a substantial uptake of magnetic label (Determine 2). Prussian blue staining revealed a homogenous ferric ion distribution in the cytoplasm, excluding the nuclei (Figures 2a,b). Prussian blue signals remained unchanged from day 2 to day 28 post simple incubation (Figures 2c,d), indicating a stable vesicular incorporation of VSOP for at least 1 month. This ratio did not differ significantly between day 2 and day 28. No apparent increase in iron-oxide particle uptake was observed upon visual inspection in lipofected cells (Figures 2e,f), which was confirmed by counting labeled cells. Overall, 96C100% of haNSCs Lobetyolin were labeled. Labeling efficacy could not be improved significantly at any time point by additional lipofection (+L) (Physique 2g), so lipofection was omitted in all further experiments. Open in a separate window Physique 2 Cytological analysis of magnetically labeled haNSCs (= 3 with 3 technical replicates each). (a) Unlabeled control cells and (b) intracytoplasmic VSOP uptake by haNSCs following incubation with 0.5 mM VSOP. (cCf) Cells were fixed with 4% phosphate-buffered saline-buffered paraformaldehyde and intracellular iron was visualized using Prussian blue staining on day 2 as shown in (c,e) and on day 28 as provided in (d,f) after labeling. (g) Cell Lobetyolin counting revealed that labeling efficacy at any time point could not be enhanced significantly by lipofection. (h) Proliferation analysis of VSOP-labeled haNSC (1.5 mM) revealed no statistically significant difference in.