Page 61 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis
P. 61

                                2. Cell migration: variations in labeled and unlabeled cell densities
The physical principle of cell migration was mimicked by the mixing of the suspensions of labeled and unlabeled cells. To characterize the effect of variations in the density of labeled cells, phantoms were created with a total of 3 x 106 cells in 200 ul 0.3% agar. The ratios of labeled and unlabeled cells were 1:1, 1:3, 1:7, 1:15, 1:31 and 1:63.
3. Intracellular variations: cell division and labeling efficiency
We generated sample series to assess the effect of heterogeneity, which may arise from uneven SPIO distribution to daughter cells or from variations in cell doubling times. Cells were harvested from an initially labeled stock at different time points after labeling. Cells were growing in cell culture, and their SPIO content was passed on to daughter cells; thus the level of cell labeling was subject to cell division.
After labeling the cells were replated and the next samples were taken at 24, 48 and 72 h. The pelleted cells were resuspended in 200 ul 0.3% agar (Becton Dickinson, Alphen aan de Rijn, The Netherlands) in a 500 ul Eppendorf tube (Fisher Scientific B.V., Landsmeer, The Netherlands).
To study the effect of variations in cell labeling, two different protocols (High and Low in the following) were followed to generate batches of labeled cells. The protocols were only different in the step preceding incubation for 24 h(see the protocol details in the ‘Cell culture and cell labeling’ section.): the added iron content was 279 ug (= 1296 ul) for the ‘High’ protocol or 233 ug (=1082 ul) for the ‘Low’ protocol. Both batches were used independently in the cell dilution series (mixing labeled and unlabeled cells) and in the cell division series.
Results
Free and incorporated labeling particles
The relaxivity showed a strong dependence on the type of the labeling particles. MPIOs exhibited the highest relaxivity r2*= 635 mM-1 s-1, followed by SPIOc r2*= 226 mM-1 s-1 and SPIO particles r2* = 170 mM-1 s-1. Compared
Quantification of iron labeled cell
59
3
 






















































































   59   60   61   62   63