Name: In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method (Video) | JoVE Journal
Uploaded: 2008-07-23T22:33:00.000+00:00
Duration: 7 min 7 s
Description: Learn the hanging drop method for in vitro differentiation of mouse embryonic stem cells into embryoid bodies. This technique enables controlled cell aggregation to study stem cell biology and advance regenerative medicine research.

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<ol>
<li>Gelatinize T75 flask using a 0.1% gelatin solution and incubate plate in a 37&deg;C, 5% CO2 tissue culture incubator overnight one day before.</li>
<li>Take out the mES cells from incubate, aspirate the medium, rinse the ES cell culture with PBS, add 0.05% trypsin solution to coat the bottom of the dish (2 ml/100mm plate).</li>
<li>Incubate at 37&deg;C for approximately 1 minutes, until the cells are sloughing off the plate. </li>
<li>Gently triturate (pipet up and down) the trypsinized cells four to six times to disperse the ES cells with a plugged Pasteur pipette,. Transfer the dispersed ES cells into a 15-ml conical centrifuge tube containing&nbsp; prewarmed (37&deg;C) mES medium. </li>
<li>Collect cells by centrifugation. Aspirate the supernatant, Add 10 ml of mES medium and pipette up and down to form an single cell suspension. </li>
<li>Transfer the cell suspension into a T75 flask pre-coated with 0.1% gelatin and incubate at 37&deg;C&nbsp; 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. </li>
<li>Spin the cells at 1000 rpm for 5 min and aspirate off the mES medium. Then add another 10 ml of mES differentiation medium and resuspend the cells by repetitive pipetting until there appears to be a fine suspension of cells.</li>
<li>Count cells with a hemocytometer use differentiation medium to dilute the stem cell suspension to a concentration of 400 to 500 cells per 20ul (20ul/drop)&nbsp; in a sterile basin.</li>
<li>Lift lid, carefully invert it and place it on top of the dish containing 10 ml of PBS.&nbsp; Using a multichannel pipette, make rows of 20ul drops on the up-turned inner surface of the lid of the tissue culture dish.</li>
<li>Carefully place the dish in the incubator for 2 days. After two days, carefully turn over the plate cover, aspirate 180 ul fresh differentiation medium and put several drops into the well of a 96-well ultralow attachment plate. Then pickup the drop with the pipette and transfer drops, one-by-one, to the 96-well plate. Place the plates into the incubator undisturbed for 3 days. </li>
<li>Coat each well of a 48-well tissue culture plate with 300ul of 0.1% gelatin. After adding the gelatin, incubate the plate overnight at 37&deg;C one day ahead before transfer the Ebs. </li>
<li>The next day, aspirate the gelatin from the 48-well plate. Then add 300 ul of differentiation medium to each well. Transfer the EBs from the 96 well plates to the 48 well gelatin-coated plates one-by-one. Change the medium the next day and then change the medium every other day to maintain the cells. </li>
<li>Spontaneous cardiaomyocyte contractions should be evident within 7 days.</li>
</ol>

<ol><li>Wobus, A. M., Wallukat, G., Hescheler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pluripotent+mouse+embryonic+stem+cells+are+able+to+differentiate+into+cardiomyocytes+expressing+chronotropic+responses+to+adrenergic+and+cholinergic+agents+and+Ca2%2B+channel+blockers%5BTitle%5D)+AND+%22Differentiation%22%5BJournal%5D)">Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers.</a> Differentiation. 48, 173-182 (1991).</li>
<li>Metzger, J. M., Lin, W. I., Samuelson, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transition+in+cardiac+contractile+sensitivity+to+calcium+during+the+in+vitro+differentiation+of+mouse+embryonic+stem+cells%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Transition in cardiac contractile sensitivity to calcium during the in vitro differentiation of mouse embryonic stem cells.</a> J Cell Biol. 126, (1994).</li>
</ol>

The disadvantages of hanging drop method are as follows: the liquid volume of a drop is limited to less than 50ul due to maintaining hanging drops on the lid by surface tension, and it&rsquo;s impossible to change the medium for hanging drops. Observation of forming EBs in drops directly with microscopy is also very difficult during cultivation. Further more , the hanging drop method consists of two steps, therefore, a series of step of the hanging drop method may be troublesome.

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in vitro differentiation of mouse embryonic stem (mes) cells using the hanging drop method

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. 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 of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

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.

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JoVE Journal

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Learn the hanging drop method for in vitro differentiation of mouse embryonic stem cells into embryoid bodies. This technique enables controlled cell aggregation to study stem cell biology and advance regenerative medicine research.

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

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