In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types in the body. Serving as a sort of repair system for the body. They can theoretically divide without limit to replenish other cells.

When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function. This promising area of science is leading scientists to investigate the possibility of cell-based therapies to treat disease in vitro. Differentiation of embryonic stem cells occurs when the cells are allowed to aggregate in suspension culture.

In the absence of mouse embryonic, fibroblast feeders and leukemia inhibitory factor, these cell aggregates are called embryo bodies. The hanging drop method is one effective procedure for the differentiation of stem cells into embryo bodies in vitro. Hi, I'm Sha Juan from the lefty of in the division of Cardiovascular Medicine at Stanford University.

Today we will show you a procedure for individual differentiation of embryonic stem cell to emry the body using the hand drop method. We typically use this method to differentiate embryonic stem cell into cardiomyocytes. This procedure, including the following steps, preparing a single cell suspension of embryonic stem cells, generating hand drop cultures, transferring hand drop cultures to ultra low attachment plates, and the plating embroid bodies onto jut coated plates.

Okay, let's get us started. The first step of the procedure is to prepare a single cell suspension of embryonic stem cells. Because we culture our undifferentiated embryonic stem cells by growing them on mouse embryonic fibroblasts, we must detach the cells and separate them from the fibroblasts.

To do this, we tryps anize the cells using standard procedures. Once the try and treatment is complete, add mouse embryonic stem cell medium to neutralize the trypsin. Next, transfer the cell suspension to a 15 mil conical tube.

Then break up the cell colonies by pipetting up and down against the bottom of the tube until you obtain a fine suspension of cells without any visible clumps. Now spin the cells at a thousand RPMs for five minutes. After spinning the cells aspirate off the supernat and resuspend the cells with 10 mils of mouse ES cell.Medium.

Transfer the cell suspension to a T 75 flask pre-coded with 0.1%gelatin and incubate at 37 degrees with 5%CO2 for one hour. After one hour, the fibroblasts have attached to the plate, but the stem cells remain in the medium. Pipette up the medium to collect the stem cells.

Spin the cells at a thousand RPMs for five minutes and aspirate off the medium. Then add another 10 mils of differentiation medium, and resuspend the cells by repetitive pipetting until there appears to be a fine suspension of cells. Count the cells using a hemo cytometer.

Now we're ready to culture the cell suspension using the hanging drop method. To begin the hanging drop culture, use differentiation median to dilute the stem cell suspension to a concentration of 500 cells per 20 microliters. Next, invert a 150 millimeter tissue culture plate cover.

Then pipette 20 microliters of the cell suspension onto the inner surface of the plate cover. Using a multi-channel pipetter, add 10 mils of PBS onto the plate. Quickly invert the plate cover and gently place it onto the plate to form the hanging drops.

Once the plate cover is in place, carefully transfer the plate to the incubator culture. The cells undisturbed for two days. Once the incubation is complete, we will proceed with transferring the hanging drop culture to ultra low attachment plates.

After two days, carefully turn over the plate cover next aspirate 180 microliters fresh differentiation medium, and put several drops into a 96 well ultra low attachment plate. Then pick up the drops with a pipette and transfer them one by one to the 96 well plate. Place the plates into the incubator undisturbed for three days.

The embryo bodies will continue growing into a ball shape. When the incubation is complete, we will be ready to plate the embryo bodies. In order to help the embryo bodies to continue to differentiate.

We transfer them to a 48 well gelatin coated plate. To do this, we first coat each well of the plate with 300 microliters of 0.1%gelatin. After adding the gelatin, incubate the plate overnight at 37 degrees Celsius.

The next day, aspirate the gelatin from the 48 well plate. Then add 300 microliters of differentiation medium to each. Well now one by one transfer the embryo bodies from the 96 well to the 48 well gelatin coated plates.

We usually change the medium the next day and then every other day to maintain the cells. We use this method to look for better protocols for differentiating embryonic stem cells. In the cardiomyocytes, we've just shown you how to perform in vitro differentiation of mouse embryonic stem cells using hanging drop cultures.

This method is effective because the round bottom of the hanging drop allows the aggregation of embryonic stem cells, thus providing a good environment for the formation of Embry bodies. When doing this procedure, it's important to remember that the number of embryonic stem cells aggregated in a hanging drop can be controlled by varying the number of cells in the initial cell suspension. That's it.

Thanks for watching. Good luck with your experiment.