Cardiac derived cells are placed onto cell culture plates for simulated ischemia. Reperfusion model ischemia is achieved with 150 minutes in a glucose free medium, while the oxygen level is below 0.5%This ischemic period leads to the shrinking and blabbing of most cells. Then normal media and oxygen levels are reintroduced to simulate reperfusion.
Mesenchymal stem cells are added and cultivated together with the damaged cells. After 24 hours of co cultivation cells are stained with calcium and Ethereum homodimer To differentiate between live and dead cells, confocal microscope, it clearly shows that two cell populations and their interactions flow cytometry analysis reveals three clusters of damaged cells, which can be plotted on a graph and analyzed statistically Stem cell transplantation. Protocols are finding their way into clinical practice, getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research.
Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate mechanisms involved in the treatment process. We designed an experimental protocol in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia, perfusion injury and after stem cell transplantation. Our design criteria, our fully in vitro system, but only the host and the donor cells are present.
So it is isolated from the surrounding tissue. Continuous measurement of cell viability before, during, and after the injury for up to 24 hours. And clear identification of both cell populations so the fate of the grafted cells can be traced.
In order to reach this goal, we set up a protocol of oxygen, glucose deprivation, which is commonly abbreviated as OGD, followed by a reoxygenation period. Our choice of host cells was age nine C two cells via known red cardio myoblast cell line. After OGD, the damaged cells were supported by transplantation of bone marrow derived mesenchymal stem cells by adding them to the culture.
This setup allowed us to investigate the intercellular connections using confocal forest and microscopy and to quantify the survival rate of damaged cells by flow cytometry. Let's get to work and see how we do it. Remove culture medium from H nine C two cells.
Wash cells twice with PBS and digest with 0.05%trypsin EDTA place Petri dishes for five minutes. In a 37 degree incubator, inhibit trypsin with DMEM culture medium. Spin down the cells at 1200 RPM for eight minutes.
After removing the super natin resuspend cells in one millimeter DM EM culture, medium count cells with he cytometer using triam blue staining C 30, 000 H nine C two cells per well. In a 12 well plate, place the plate into the 37 degree carbon dioxide incubator for 24 hours. Remove medium by aspiration wash cells twice with PBS.
Add three millimeters glucose free medium. Place the plate into the cell incubation system. Start nitrogen gas to purge the oxygen out of the incubation system.
Wait until the level of oxygen reaches 0.5%Then start the timer, 0.5%Oxygen means approximately three millimeters of mercury partial tension subject cells to 150 minutes of simulated ischemia. During ischemia, cells are getting detached from the surface and their membrane starts blabbing. We found that each population needs slightly different lengths of time, anti reaching a 60 to 80%of cell image.
That's why we suggest to begin with the two R incubation. And if it's not enough, increase the time of OGD. In our case, age nine C two cells typically need 150 to 70 minutes of OGD At the end of simulated ischemia.
Remove the glucose free medium from the cells pipette normal culture medium to the wells. Place a 12 well plate for 30 minutes in the 37 degree carbon dioxide incubator. This is the reperfusion period.
During simulated ischemia, prepare rescue cells for transplantation. Dilute the fluorescent diet, vibrant DID in one to 200 ratio in DMEM culture. Medium aspirate medium from the rescue cells and add 300 microliters DMEM from vibrant Brent Place Petri dishes for 30 minutes in the 37 degree carbon dioxide incubator.
After 30 minutes at the end of the reperfusion at thousand mesenchymal stem cells per well to the post ischemic H nine C two cardiac myoblasts. The initial steps are the same as H nine C two seeding washing two times with PBS trypsin and counting. Place co culture of cells into the 37 degree carbon dioxide incubator for 24 hours.
After 24 hours. Harvest a culture media and the adherence cells from wells with ization. It is very important to collect a culture medium also because some dead cells could have a detached from the cultured dish surface during the 24 hour incubation.
Suspend the cells in 500 microliter live, dead, die solution, five NMO calcium, and 400 NMO Ethereum homodimer in PBS five times 10 to the power of four cell per milliliters. In case of co cultures, mesenchymal stem cells are distinguished from H nine C two cells by the respective fluorescent cell labeling. This way it is possible to detect cell fusion and cell to cell contact sites.
The ratio of dead cells can be evaluated in four independent fields of view, using a 10 times objective for each culture in a blind fashion, which gives a reproducible cell count result. Transfer cells to flow cytometric tubes. Start the cell Quest Pro software to set up the flow cytometer for measurement.
Prepare control samples of live and dead cells. Living cells are cultured at the same density in normal conditions and stain with calcium. Dead cells are prepared with one hour treatment with 10 micromole hydrogen peroxide stained with Ethereum, homodimer, and harvested in the same manner the instrument settings should be adjusted so the living cells are calcium positive and Ethereum homodimer negative.
While the dead cells are calcium negative and Ethereum homodimer positive, the percentage of these populations can be expressed as bars on a graph and statistical analysis can be performed On these values. We found that the addition of healthy stem cells increase the ratio of surviving cells in the population. Interestingly, we also observed that the cells which were saved after the injury can be gated out from those which were dead, but also from those that remained alive.
We believe that this population warrants further investigations. After watching this video, you should have a good understanding of how to conduct simulated ischemia experiments with in vitro stem cell transplantation in a standardized way. So that's it.
Thanks for watching and good luck with your experiments. In case you have any technical questions, do not hesitate to contact us. We'll be there to help out.
