Name: in vitro differentiation of mouse embryonic stem (mes) cells using the hanging drop method
Uploaded: 2008-07-23T22:33:00
Description: Watch this Scientific Journal Video about In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method at JoVE.com

<ol>
<li>Gelatinize T75 flask using a 0.1% gelatin solution and incubate plate in a 37&deg;C, 5% CO<sub>2</sub> 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.<br /></li>
<li>Gently triturate (pipet up and down) the trypsinized cells four to six times to disper...

<ol>
	<li>Wobus, A. M., Wallukat, G., Hescheler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+mouse+embryonic+stem+cells+are+able+to+differentiate+into+cardiomyocytes+expressing+chronotropic+responses+to+adrenergic+and+cholinergic+agents+and+Ca2++channel+blockers.">Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers.</a> <em style='font-style: italic'>Differentiation</em>. <b>48</b>, 173-182 (1991).</li><li>Metzger, J. M., Lin, W. I., Samuelson, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transition+in+cardiac+contractile+sensitivity+to+calcium+during+the+in+vitro+differentiation+of+mouse+embryonic+stem+cells.">Transition in cardiac contractile sensitivity to calcium during the in vitro differentiation of mouse embryonic stem cells.</a> <em style='font-style: italic'>J Cell Biol</em>. <b>126</b>, (1994).</li></ol>

<p class="jove_content">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.</p>

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<td>leukemia inhibitory factor(LIF)</td>
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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. 

Watch this Scientific Journal Video about In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method at JoVE.com

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

<p class="jove_content">This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.</p>

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 ...

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