The goal of the presentation is to demonstrate an easy and replicable method of MASI production with para oxides labeled HMEs for tracking of stem cell based therapy. With MR Imaging labeling, media is prepared added to a T 75 flask and incubated for four hours. Then cells are tryps, inized and washed.
Three times after washing cells are resuspended and agros gel is added to the tube. Now the cells are ready for implantation into cartilage defects. Hi, I'm Alex.
I need re peel from the laboratory of High Delta Blink, the Contrast Media Laboratory in the Department of Radiology at the University of California San Francisco. I'm Lydia Men. So also from the Contrast Media Lab.
Today we'll show you a procedure for labeling medicine hemo stem cells with iron oxide nanoparticles generating cell agros constructs, and implanting these constructs into cartilage defects. We use this procedure in our laboratory to track mesenchymal stem cells non-invasively with magnetic resonance imaging. So let's get started.
To begin, cells are grown to 80%confluence at least 18 hours before labeling. During this time, prepare labeling media so that it will be ready for use when the cells are confluent. To do this add end durum to the sample at a dose of 100 micrograms of iron per milliliter of serum free media.
After the cells are grown to confluence, aspirate the culture media and wash the cells with PBS or serum free media. Next, aspirate the rinse solution and add the prepared labeling media. Incubate the cells at 37 degrees Celsius, 5%carbon dioxide for four hours.
After the four hour incubation, aspirate the labeling media. Rinse the cells with PBS and then trypsin ice. Once tryis, wash the cells three times with PBS and centrifuge them at 400 RCF for five minutes.
After this washing step, resus, suspend the cells, count them, assess them for viability, and finally use them as needed for experimentation. Now let's see how to create matrix associated stem cell implants. Begin by preparing a fresh agro solution.
Once the agros is cooled down to 42 degrees Celsius, count the cells and centrifuge them in a 1.5 milliliter einor tube. Following centrifugation, discard the supernatant and mix the cells with dobe echo's modified eagles medium to reach a concentration of 30 times 10 to the six cells per mil before mixing the cells with the warm aros preheat the pipette tip by pipetting hot, sterile PBS up and down. This will prevent agros from solidifying inside the tip.
Then take up the same volume of aros as media and mix it thoroughly with the cell media suspension and pay attention that no bubbles are created. Keep the einor tube inside a preheated water bath while mixing so that the agros does not cool down enough to solidify when the suspension is homogenous. The cells are implanted in the defect and are now ready for MR.Imaging.
Now we'll show you some representative results, first of our labeling technique and second of our implantation technique. After labeling, the cells in the flasks still have the same appearance as the unlabeled cells. As expected, the viability of labeled cells is unchanged as demonstrated by the Caspase three test.
The combination of labeled cells with agros leads to a signal loss in the implants as seen in T two weighted MR images, non labeled cells don't show any signal. Decrease sections of cell agros constructs stain with Prussian blue. Confirm the intracellular iron accumulation.
We've just shown you how to label mechy stem cells with the iron oxide nanoparticle end interim. How to create an agros constructs with the label cells and how to implant them into cartilage defects. When doing this procedure, it's important to remember to prewarm your pipette tip prior to adding warm aros to the cell media suspension.
It is also crucial for Mr.Imaging to avoid formation of any bubbles. So that's it. Thanks for watching and good luck with your experiments.
