Welcome to the laboratory of Dr.Haiku Dal Link. We would like to thank the California Institute of Regenerative Medicine for funding this project for the evaluation of new stem cell therapies. It is important to non-invasively track the injected cells in vivo.
This is possible by labeling the cells in vitro with specific or multifunctional contrast agents for MR imaging or optical imaging. This video will show you how to label human mesenchymal and human embryonic stem cells for subsequent in vivo imaging. Hi, I'm Tobias Henning from the Contrast Agent Research Group at the Center for Molecular and Functional Imaging in the Department of Radiology at the University of California San Francisco.
Today I'm going to show you procedures for in vitro labeling of human mesenchymal stem cells with peric, carone, and of human embryonic stem cells with phem oxides. This technique is useful in order to localize injected cells in vivo or to obtain information about their functional state. The procedures for cell labeling with MRI conscious agent varies for embryonic and mesenchymal stem cells, but both techniques involve the following steps, preparing the cell culture that is plating of cells as adhere at cell cultures preparation of labeling, media labeling of the cells by simple incubation trypsin of stem cells and washing off free conscious agent evaluation of labeling efficiency and cell viability.
So let's get started and label some cells. Now let's begin with labeling mesenchymal stem cells with Theo Carbatrol. First, I will show the technique for labeling human mesenchymal stem cells with theo carron and iron oxide based contrast agent for MR Imaging.
To begin, we plate the cells 18 to 24 hours before the labeling procedure in T 75 flasks at a confluence of 80%Otherwise, if you use a different culture dish that equals to about 10, 000 cells per square centimeter the next day, we start with preparing the labeling media by adding 30 microliters of reservist to eight milliliters of serum free media. This will label one T 75 FLA at a co fluency of 80%and corresponds to a concentration of a hundred micrograms of iron per milliliter of media. Now we take off the culture media and wash the cells once with PBS or serum free media.
We do this to get rid of residual serum proteins and other constituents of the media that could bind the contrast agents and influence labeling efficiency. Add the labeling media to the flask and put the flask back into the incubator. After two hours, add two ml of FCS so that a final concentration of 20%of FCS is achieved.
We do this to ensure that the cells do not receive any stimulus to differentiate that it's dead grow in their familiar environment. Now we incubate the cells for 18 hours. The next day we rinse the cells with PBS and then gize them according to standards culture protocols in order to get rid of free contrast agent.
Once Tryps inized, the cell suspension is washed three times by centrifuging at 400 RCF for five minutes, and reus suspending in PBS. That's it. The final cell P can be resuspended and is ready for further experiments.
We count the cells now as we might have lost some during the previous washing steps. We also perform viability testing at this point using the trippin blue exclusion assay and take some samples for spectrometric analysis to measure the labeling efficiency. Now let me show you how to label human embryonic stem cells with phem oxides.
To begin, we plate the human embryonic stem cells in 10 centimeter dishes. As usual, these dishes are pre-coded with gelatin and have irradiated feeder cells on them. You can use this protocol for feeder free cultures as well.
We let the human embryonic stem cells attach and grow for approximately three to four days so that they form medium sized colonies. During this time, we ensure that the colonies do not grow too big because larger colonies are more difficult to break up. Later, differentiated cells can contaminate these cultures.
So depending on the cleanliness of the colonies, we may have to get rid of differentiated cultures by dissection. Now we prepare the labeling media that consists of theorem oxides at a concentration of a hundred micrograms iron per milliliter and full human embryonic stem cell media. For this, we mix 89 microliters, phem oxides, and 10 milliliters of full growth media.
As for mesenchymal stem cells, we wash the dish once with complete media to get rid of dead cells or debris. Then we add 10 milliliters of labeling media per dish and incubate the cells for four hours. Now the labeling is complete.
In the next step, we need to wash the cells and get a single cell suspension. For further use For this, we first rinse the dish with PBS. We now replace the PBS with five milliliters of 0.25%trypsin and incubate for five minutes in the incubator or until the cells detach.
It helps to tap the dish frequently the cells have now detached. Keep in mind that if the dish contained larger colonies, that might be some clumps left to get rid of these clumps. We mix the whole suspension thoroughly by pipetting up and down, using a serological pipette, and then let it sit for another two minutes.
In the trypsin. We do not use an eppendorf pipette as repeated pipetting of the cells through the thin tip can break the cell membranes if everything worked right. We have a single cell suspension now that is mixed with a lot of debris originating from the gelatin coating the embryonic stem cell matrix and dead cells.
We can now get rid of remaining clumps or complexes by passing the cells through a 40 micrometer cell strainer. Now we need to isolate the human embryonic stem cells from the irradiated feeder cells. This can be done efficiently by plating the cell suspension on a gelatin coated dish.
If we do not manipulate the dish, all cells will sediment down, but the feeders will attach faster than the human embryonic stem cells. So if we take off the SUP natin after 45 minutes, it should contain mainly human embryonic stem cells, while the feeders should mainly be attached to the dish. This way, we obtain a single cell suspension of magnetically labeled human embryonic stem cells that can be used for further experiments as in the previous protocol.
At this point, you need to count the cells and take samples for viability assessment and measuring of labeling efficiency. So this finishes up the protocols I wanted to show you today. Now let's take a look at how we can track labeled stem cells in vivo.
This slide shows OC Carone labeled stem cells. After they have been injected into the brain of a mouse, we resus suspended 20, 000 cells in the volume of 75 nanoliters and injected them into the sub ventricular zone. The iron oxide in the implanted cells causes a susceptibility artifact that can be seen on MR.Images.
We've just shown you how to label embryonic and mesenchymal stem cells. In vitro with Mr.Contrast agents tracking mesenchymal and embryonic stem cells in vivo has enormous potential for evaluating new stem cell therapies. So that's it.
Thanks for watching and good luck with your cell labeling.
