Mesenchymal stem cell labeling and in vitro MR characterization at 1.5 T of new SPIO contrast agent: Molday ION Rhodamine-B™

Abstract

In vivo detection of transplanted stem cells is requisite for improving stem cell-based treatments by developing a thorough understanding of their therapeutic mechanisms. MRI tracking of magnetically labeled cells is non-invasive and is suitable for longitudinal studies. Molday ION Rhodamine-B™ (MIRB) is a new superparamagnetic iron oxide (SPIO) contrast agent specifically formulated for cell labeling and is readily internalized by non-phagocytic cells. This investigation characterizes mesenchymal stem cell (MSC) labeling and MR imaging properties of this new SPIO agent. Effects of MIRB on MSC viability and differentiation as well as cellular loading properties were assessed for MSC labeled with MIRB at concentrations from 5 to 100 µg Fe/ml. Labeled MSC were evaluated, in vitro, on a clinical 1.5 T MRI. Optimal scanning sequences and imaging parameters were determined based on contrast-to-noise ratio and contrast modulation. Relaxation rates (1/T(2)*) for gradient-echo sequences were approximated and an idealized limit of detection was established. MIRB labeling did not affect MSC viability or the ability to differentiate into either bone or fat. Labeling efficiency was found to be approximately 95% for labeling concentrations at or above 20 µg Fe/ml. Average MIRB per MSC ranged from 0.7 pg Fe for labeling MIRB concentration of 5 µg Fe/ml and asymptotically approached a value of 20-25 pg Fe/MSC as labeling concentration increased to 100 µg Fe/ml. MRI analysis of MIRB MSC revealed long echo time, gradient echo sequences to provide the most sensitivity. Limit of detection for gradient echo sequences was determined to be less than 1000 MSC, with approximately 15 pg Fe/MSC (labeled at 20 µg Fe/ml). These investigations have laid the groundwork and established feasibility for the use of this contrast agent for in vivo MRI detection of MSC. Properties evaluated in this study will be used as a reference for tracking labeled MSC for in vivo studies.

Figure 1

Light (top row) and flourescence (bottom row) microscopy images of control unlabeled MSC (left column) and MIRB labeled MSC (right column). Cells are stained with Prussian Blue for Fe prescence (400× magnification).

Figure 2

Flow cytometry data for viability (7AAD-) and labeling efficiency (Rh-B+) as a function of MIRB labeling concentration.

Figure 3

Average internalized FE per MSC as a function of MIRB labeling concentration with asymptotic curve fit. Error bars represent 15% approximated error from cell counting.

Figure 4

(top) Gene expression data for control (C) and MIRB labeled (L) MSC from three experiments. (bottom) Representative flow cytometry data for labeling efficiency, viability and phenotype for control (grey stripes bar) and MIRB labeled (black bar) MSC.

Figure 5

Relative proliferation rates for PHA stimulated PBMC with the addition of no MSC, control MSC or MIRB labeled (20 μg Fe/ml) MSC (n =1 experiment).

Figure 6

Light microscopy images of unlabeled and labeled (20 μg Fe/ml) MSC differentiation into fat (top) and bone (bottom). Control cells (both labeled and unlabeled) were not provided with adipogenic or osteogenic media.

Figure 7

CNR vs TE for spin echo (top) and gradient echo (bottom) sequences for labeled and unlabeled MSC pallets with cell count of 1E5. Data shown as mean ± SEM for 6 aquisitions.

Figure 8

Optimized CNR for each of the considered imaging sequences vs. MSC concentration (top) and MSC pellet count (bottom). Mean ± SEM for 4 acquisitions.

Figure 9

CNR for SE and GE sequences for MSC pellets labeled at increasing MIRB concentrations (top left) and GE (500/60) image of phantom used with 5E4 MSC labeled with MIRB in concentrations (from left to right) of 50, 40, 30, 20, 10 μg Fe/ml (top right). CNR vs. MSC pellet cell count for MSC labeled with 20 μg Fe/ml (lower left) and GE (500/60) image of phantom set used with (from left to right) 1.25E4, 2.5E4, 3.75E4, 6.25E4,1.2E5, 2.5E5 and 5E5 MSC labeled with 20 μg Fe/ml (lower right).

Figure 10

MOC (top) and CNR (bottom) curves vs. TE for GE (TR =500) sequences for labeled and unlabeled MSC. Mean ± SEM for 4 acquisitions.

Figure 11

T2* curves as a function on cell count (top left) and MIRB labeling concentration (bottom left) and T2* map of phantom with various MIRB MSC pallets (right). Mean ± SEM for 6 acquisitions.

Figure 12

GE (500/60) image of phantom used for limit detection. From left MIRB MSC pellet counts of 5E3, 2E3, 1E3, 1E3, 1E3.

Figure 13

Optimal MIRB labeling concentration. Relative CNR, Labeling efficiency, Fe/MSC & viability as a function of MIRB labeling concentration.