Magnetic Resonance Imaging or MRI has emerged as a predominant imaging modality to localized transplanted stem cells and to better understand the mechanism of regeneration. These MRI studies typically use iron oxide nanoparticles as the imaging contrast agent. However, iron particles can remain in the tissue after stem cell death and can be taken up by macrophages resulting in suboptimal conditions for tracking the viable transplanted cells.
This video will demonstrate that manganese chloride is an effective alternative. Contrast agent labeling with manganese chloride offers several advantages over iron oxide labeling. For example, manganese gives a positive signal, whereas a negative signal is produced by ion particles.
In addition, manganese ion is known to enter cells via voltage gated calcium channels. So its appearance in MRI indicates viable by biologically active cells. Hi, I'm Uni mother from the laboratory of Philip Yang in the division of Cardiovascular medicine at Stanford University.
Today I will show you how to label human embryonic stem cells with manganese for magnetic resonance imaging. This procedure includes the following steps, labeling human embryonic stem cells with contrast agents performing in vitro cellular magnetic resonance imaging and analyzing the MRI results. So let's get started.
The first step in the labeling procedure is to prepare human embryonic stem cells, which have been cultured in fetal free conditions. To do this, trypsin the cells for five minutes, then neutralize the trypsin by adding culture media. Next, spin down the cells by centrifuging at 800 RRP M for five minutes at 20 degrees Celsius.
After Resus suspending the cell palette in PBS, count the number of cells by trip and blue staining based on the cell count obtained, divide the cells into the required number of quats into conical tubes. In this case, I'm dividing the cells into four Ts of 3 million cells. Palate the cell again by centrifugation just before starting the manganese labeling dissolve fresh manganese chloride in 0.9%sodium chloride to make a 0.1 millimolar manganese chloride solution.
In order to observe calcium channel activity, we prepared for samples. The first sample is our control, which contains cells incubated in 0.9%sodium chloride alone. The second sample contains cells incubated in 0.1 millimolar manganese chloride samples three and four contain cells incubated in 0.1 millimolar manganese chloride with either five micromolar of the calcium channel agonist AK 8 6 44 or 250 micromolar of the calcium channel antagonist.
The peril next incubate the four samples at 37 degrees Celsius with 5%CO2 for 30 minutes After 30 minutes, spin down the cells at 800 RPM for five minutes at 20 degrees Celsius. Then aspirate the supan agent and wash the labeled cells twice with PBS. After washing reus, suspend each pallet with 200 microliter PBS and transfer into PCR tubes.
These pallets are usually white. Okay, we are done preparing the manganese labeled cells for comparison purposes. We will also prepare cells using the more traditional iron labeling methods to begin the iron labeling.
Nick's clinical grade protamine sulfate with distilled water to get a one milligram per milliliter stock solution. Next in the tube containing human embryonic stem cell culture media, add film oxide at a hundred microgram per milliliter combined with the protamine sulfate at 12 microgram per milliliter. Then mix the solution for five minutes.
After mixing the solution, add it to your cells using an equal volume of the mixed solution and the cell culture media to achieve a final concentration of 50 microgram per milliliter of oxide and six microgram per milliliter of the protamine sulfate. Incubate cells for 12 hours after 12 hours, wash the cells twice with PBS with the last wash containing 10 unit per milliliter heparin to dissolve the extracellular oxide protein sulfate complex. After washing trypsin cells to obtain a single cell suspension, then count the cells and qua into the required number of samples.
In this case, I am preparing one sample with 1 million cells and the other with 3 million cells. Next P cells, these palettes should be dug orange, brown in color. Finally, we resus suspend each pallet with 200 myta of PBS and transfer into PCL tubes.
Okay, we are done labeling the cells. Let's proceed with the cellular MR imaging. To begin the imaging, we first make a phantom in order to stabilize the tubes containing the cell palettes and also to avoid artifacts from the surrounding air during the scanning.
To do so, mix 1%of copper sulfate and 0.8%of agar in distilled water and bring it to a boil by microwaving for five to seven minutes. This mixture is then cooled for at least one hour to attain a gel. Next, the tubes containing the label cell pellets are placed in the phantom for scanning.
In vitro cellular MRI is performed with a 3.0 T scanner using a clinical knee coil. We use the Signa made by GE Medical Systems scan the manganese labeled cells for each of the four samples using a spin echo sequence. Parameters for the sequence have been worked out from previously studied T mapping of the labeled cells.
We use echo time at minimum a repetition time of 800 millisecond field of view of 12 by 12 centimeter matrix 2 56 by 2 56. Next one. Next, scan the iron labeled cells by using a gradient echo sequence.
We use the following parameters for this imaging. Echo time of 10 milliseconds, a repetition time of a hundred milliseconds. Flip angle of 30 degrees, field of view of 12 by 12 centimeter matrix 2 56 by 2 56 and next one.
Okay, we are all done with the imaging. Now let's take a look at the results. Here are the MRI scanning results for four different samples of manganese labeled cells.
Sample number one is our control, which contains cells incubated in 0.9%sodium chloride alone. Sample number two contain cells incubated in 0.1 millimolar manganese chloride. Sample number three contain cells incubated in 0.1 millimolar manganese chloride with five micro of the calcium channel agonist vacay 8 6 4 4.
Sample number four contains cells incubated in 0.1 million MO manganese chloride with 250 micro moura of the calcium channel antagonist veil. Compared to the cells labeled with manganese alone, the signal intensity increases in the presence of the calcium channel agonist and decreases in the presence of calcium channel antagonist. The area of signal increase for manganese labeled cells are measured by using Image J.This results indicate the manganese does enter via the voltage gated calcium channel.
Now let's compare this results with results from the iron labeled cells. Note the difference between the bright positive signal from manganese labeled cells versus the dark negative signal from iron labeled cells. We've just shown you how to perform manganese labeling of human embryonic stem cells for in vitro cellular magnetic resonance imaging.
This procedure is an effective method to track stem cells because one manganese can be easily introduced into cells via a simple 30 minute incubation. Two, manganese labeled cells provide clear images with a positive signal and three manganese enter cells via the voltage gated calcium channel as shown by the use of calcium channel agonists and antagonists, and thus only enters the viable cells. Therefore, we believe that manganese labeling of human embryonic stem cells has great potential for precise in vivo cellular MRI to track the effect of transplanted human embryonic stem cells into ischemic injured areas.
So that's it. Thanks for watching and good luck with your experiments.
