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

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1Division of Cardiovascular Medicine, Stanford University

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

    Date Published: 7/23/2008, Issue 17; doi: 10.3791/825

    Cite this Article

    Wang, X., Yang, P. In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method. J. Vis. Exp. (17), e825, doi:10.3791/825 (2008).


    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.


    1. Gelatinize T75 flask using a 0.1% gelatin solution and incubate plate in a 37°C, 5% CO2 tissue culture incubator overnight one day before.
    2. 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).
    3. Incubate at 37°C for approximately 1 minutes, until the cells are sloughing off the plate.
    4. 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  prewarmed (37°C) mES medium.
    5. Collect cells by centrifugation Aspirate the supernatant, Add 10 ml of mES medium and pipette up and down to form an single cell suspension.
    6. Transfer the cell suspension into a T75 flask pre-coated with 0.1% gelatin and incubate at 37°C  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.

    7. 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.
    8. 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)  in a sterile basin.
    9. Lift lid, carefully invert it and place it on top of the dish containing 10 ml of PBS.  Using a multichannel pipette, make rows of 20ul drops on the up-turned inner surface of the lid of the tissue culture dish.
    10. 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.
    11. 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°C one day ahead before transfer the Ebs.
    12. 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.
    13. Spontaneous cardiaomyocyte contractions should be evident within 7 days.

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    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’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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    Name Type Company Catalog Number Comments
    DMEM Reagent GIBCO, by Life Technologies 11965-092
    Fetal Bovine Serum(FBS) Reagent Hyclone SH30070.03
    L-Glutamine Reagent GIBCO, by Life Technologies 25030
    Non-Essential Amino Acids Reagent GIBCO, by Life Technologies 11140-050
    Penicllin / Streptomycin Reagent GIBCO, by Life Technologies 15140-122
    Sodium Pyruvate Reagent GIBCO, by Life Technologies 11360-70
    β-mercapt–thanol Reagent Chemicon International ES-007-E
    leukemia inhibitory factor(LIF) Reagent Chemicon International LIF2005
    96-well ultralow attachment plate Tool Corning 3474




    Dear Doctor Xiang Wang,  your technique appear useful and very interesting. I' m studying mES and I want to differentiate them into simple embryoid bodies, but not into predefinite cells. So I have a question for you. The differentiation medium could be a stem cell medium just without LIF? Or do I need to add some elements to my medium to produce embryoid bodies? Thank you very much and best regards,  Barbara Tondelli.

    Posted by: AnonymousOctober 8, 2008, 7:13 AM

    hi, i work together w/ xiang. he returned to china so i asked him to respond to you but in case he dŒsn't, let me answer: 1. yes. embryoid bodies can be made w/ the stem cell medium w/o lif. you need to create a voumetric clusters. this could be done using the hanging drop method or plate in cluster of cells. we have had best exerience making hanging drop. hope this helps.   phil

    Posted by: AnonymousOctober 8, 2008, 12:09 PM

    Hello I want to know the function and concentration here of sodium pyvurate. Can the heartbeat appear in a differentiation medium without sodium pyvurate? And if the plates are taken out sometimes during the suspension culture period, dŒs it matter so much? I've been trying but no cardiocytes seemed to appear, and I cannot find the key to this. Thank you a lot!  

    Posted by: AnonymousNovember 7, 2008, 3:02 AM

    Dear Huijun Zhu,   I use 1mM Sodium pyvurate for mESC culturing. Sodium pyvurate works mostly  as  an additional source  energy. It also have protective effects against hydrogen peroxide.  According to my experience,  Sodium pyruvate is not necessary for differentiation. It 's not good for moving  out of the incubate when the EBs are in hanging drop situation. It should be fine to take out the plate carefully and take a simple look once they were in ultralow attachment plates. Hope this helps, Good luck!   Xiang Wang  

    Posted by: AnonymousNovember 10, 2008, 8:00 AM

    Thank you Xiang Wang! The cardiaomyocyte contractions became obvious after 9days. But the contraction of a certain area became weaker and finally disappeared. Is it  normal or something gŒs wrong? And how can I purify the cardiaomyocytes? How can I increase the percent of the cardiomyocytes? Thank you a lot!

    Posted by: AnonymousNovember 18, 2008, 3:59 AM

    Dear Doctor Xiang Wang

    is necessary to add gelatin on the plate or not??

    Posted by: AnonymousJune 12, 2009, 10:42 AM

    Dear Doctor Xiang Wang,

    is it possible to split the cardiomyocytes? Or how can i isolate them, to start my assays with them?
    Thank you very much.

    Kind regards

    Posted by: AnonymousApril 26, 2011, 11:27 AM

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