Welcome to the laboratory of Dr.Hiku Alge Blink. We would like to thank the California Institute of Regenerative Medicine for funding this project. Labeling stem cells with fluorescent contrast agent allows for an in vivo tracking of transplanted stem cells in vivo.
Subsequent optical imaging of the labeled stem cells permits longitudinal studies where one is capable of non-invasively monitoring the migration of the cells in vivo. This video will show you how to label both human embryonic stem cells and human mesenchymal stem cells with fluorescent dyes. Hi, I'm Sophie Boddington from the Contrast Agent Research Group in the Department of Radiology at the University of California at San Francisco.
Today I would like to show you two different techniques for labeling stem cells with fluorescent contrast agents. One for labeling mesenchymal stem cells with the fluorescent dye, DID, and another for labeling human embryonic stem cells with endign ingra. So let me start by showing you our technique for labeling human mesenchymal stem cells with DID.
To begin the procedure for labeling mesenchymal stem cells, we have to trys anize and count them to get a suspension with defined number of cells. Since this is standard tissue culture technique, we will not show it in detail. We want to suspend the cells that we'll label at a density of 1 million cells per milliliter in serum free culture medium.
First, we add five microliters of DID contrast agent per milliliter of cell suspension. Now we mix the solution by gentle pipetting cells are incubated with labeling solution in a six well low attachment dish at 37 degrees Celsius for 20 minutes. Once the simple incubation is complete, we must now transfer the cell solution to a 15 milliliter centrifuge tube.
We centrifuge it down at 400 RCF for five minutes. Next, we aspirate out the labeling media ensuring not to disturb the pellet. We now wash the cells with PBS.
We pipette the cells up and down, making sure to break up the cell palette. The latter two steps. We repeat two more times so that we have a total of three washing steps.
Now we count the cells and perform a trip and blue test to determine the cell viability. After determining cell viability, your cells are now ready to be imaged in the optical imager. To begin the procedure for labeling human embryonic stem cells, we must first prepare the contrast agent known as Indocyanine Green.
Then we mix it with the transfection agent known as protamine. To begin, we measure out one milligram of the indocyanine green powder and dissolve it in a hundred microliters of DMSO. We add 400 microliters of DMEM containing 10%FCS to the mixture and shake it well, which leaves us with a concentration of two milligrams per milliliter of indocyanine green.
Next, we add the transfection agent protamine, which acts as a shuttle for the contrast agent, allowing it to enter the cell more efficiently. Now we mix five microliters of protamine sulfate at 10 milligrams per milliliter with 300 microliters ICG and 300 microliters of serum free DMEM. Now we shake the new transfect solution for five minutes to allow complexes to form.
Next, we aspirate out the old media and add 10 milliliters of PREWARM DMEM to the cells. We add the previously prepared protamine ICG solution to the cells and start the one hour incubation by placing the dish in the incubator at 37 degrees Celsius. After the incubation is complete, we remove the dish from the incubator, aspirate out the labeling solution and wash the cells by rinsing the dish with five milliliters of PBS.
The cells are then tryps inized for five minutes at 37 degrees Celsius. Shaking the dish will help to lift the cells into suspension. Now we gently pipette up and down to break up the remaining colonies.
We neutralize the trypsin by adding an equal amount of DMEM containing 10%FCS to the dish. Next, we transfer the cell solution to a 15 milliliter tube and centrifuge the solution at 400 RCF For five minutes. We resuspend the cells in full media.
We can now get rid of residual clumps or complexes by passing the cells through a 40 micrometer cell strainer. Once we have a clump free cell solution, we aspirate out the old media and resuspend the pellet in pre wormed full human embryonic stem cell media. At this point, we need to separate the mouse feeder cells from the human embryonic stem cells.
This is done to ensure that later we are only imaging the stem cells. For this, we transfer the cell solution to a gelatin coated 10 centimeter Petri dish. We put the dish into the 37 degree incubator and let it sit for 45 minutes.
During this time, make sure not to disturb the dish. Now the feeders will adhere to the dish and the stem cells will not. We transfer the solution out of the Petri dish and now we have a labeled single cell solution of human embryonic stem cells.
We can now count the cells and perform a trip in blue test on them. After confirming viability, our cells are ready to be imaged. The slide shows an example of how stem cells can be visualized in vivo in mice after they have been tagged with fluorescent contrast agent and administered through an IP injection.
Serial optical imaging scans shown initial accumulation of the cells in the lungs, followed by redistribution in the liver, bone marrow of the extremities and the thyroid gland. The fluorescent label can also be used for direct depiction of transplanted stem cells in histopathology specimens with fluorescent microscopy. We've just shown you how to label stem cells with fluorescent contrast agents when performing cell labeling.
It's important to remember to standardize every step. We do this to ensure a consistent labeling efficiency and cell viability so that results from different experiments can be compared. So that's it.
Thanks for watching and good luck with your cell labeling.
