In this technique. In translational science cells are labeled using an FDA approved ultra-small super paramagnetic iron oxide called far amatol. Begin with human mesenchymal stem cells plated at 80%con fluency C, then transfect the contrast agent far amatol in serum free media to label the cells continue to harvest and enumerate the labeled H HMCs for experimental use.
Ultimately, magnetic resonance imaging results can show a significant negative contrast or T two effect from U-S-P-I-O labeled cells. Welcome to the DDR link lab at the Molecular Imaging Program at Stanford. The implications of this technique extends towards in vivo tracking of stem cell implants through direct visualization by non-invasive MR Imaging.
The main advantage of this technique over existing techniques like labeling with S PIOs is that the U-S-P-I-O OL is the only FD approved iron oxide nanoparticle available that may be used for cell labeling by an off-label use In a 15 milliliter conical tube. Dilute fair ictal in one milliliter of serum free media in a second tube dilute protamine sulfate in one milliliter of serum free media, equilibrate both solutions at room temperature for five minutes. Now combine the solutions and let rest for five minutes to permit U-S-P-I-O protamine sulfate complex formation.
Then add five milliliters of serum free media to create a nanoparticle labeling solution. Grow a confluent culture of human mesenchymal stem cells that were plated at 80%co fluency at least 18 to 24 hours prior to labeling. Aspirate the media from cells.
Gently wash cells with three milliliters of prewarm serum free media to remove potential impairments to contrast agent uptake and labeling efficiency. Now add seven milliliters of labeling solution and place the cells into an incubator for four hours. After four hours, remove the labeled cells from incubator and add 700 microliters of fetal calf serum, then incubate for an additional 20 hours.
Rinse the cells with prewarm magnesium, calcium free phosphate buffered saline. Then add two milliliters of PREWARM, 0.05%trypsin, and tilt the flask back and forth to ensure the entire surface of the flask is covered. Place the cells in an incubator for five minutes or until cells begin to detach from the plate if necessary.
Confirmed attachment under a microscope and gently tap the sides of the flask to facilitate cell surface detachment. Now add four milliliters of prewarm complete media. Gently rinse the flask and transfer the entire solution In a 15 milliliter conical tube, harvest the cells by centrifugation wash pellets three times in five milliliters of media.
Finally, enumerate viable cells. The cells are now ready for analysis. These MRI show sagittal cross-sections through pellets of human embryonic kidney cells in einor tubes compared to the unlabeled controls in tubes.
A pH amatol labeled stem cells demonstrate a significant darkening or negative contrast effect on T two weighted MR images and a brightening or positive contrast effect on T one weighted MR images. This iron oxide label is a functional probe in several embryonic stem cell lines. These dose curves demonstrate that as little as 10, 000 farol labeled cells can be detected via T two weighted MRI.
The labeled stem cells can be successfully differentiated into various cell types or intravenously injected for in vivo investigation. For example, these MRI visualizations indicate oxit labeled stem cells injected into mirroring cerebral ventricles as shown by the arrows. While attempting this procedure, it's important to remember to rinse the cells three times with PBS as shown in the video to remove residual free contrast agent on the surface of the cells that can lead to an overestimation of the contrast effect of labeled cells After its development.
This technique will pave the way of researchers in the field of regenerative medicine to explore further the clinical potential of in vivo stem cell engraftment with long-term visualization and stem cell tracking.
