In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method
Xiang Wang, Phillip Yang
Division of Cardiovascular Medicine, Stanford University
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0:00 Title0 0:21 Subscription Lock21 1:10 Introduction70 2:06 Preparing Single Cell Suspension of Embryonic Stem Cells126 3:46 Generating Hanging Drop Cultures226 4:39 Transferring Hanging Drops to Ultralow Attachment Plates279 5:17 Plating Embryoid Bodies onto Gelatin- Coated Plates317 6:12 Conclusion372
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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.
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- 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.
- 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).
- Incubate at 37°C for approximately 1 minutes, until the cells are sloughing off the plate.
- 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.
- Collect cells by centrifugation. Aspirate the supernatant, Add 10 ml of mES medium and pipette up and down to form an single cell suspension.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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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| DMEM |
Reagent |
Gibco |
11965-092 |
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| Fetal Bovine Serum(FBS) |
Reagent |
Hyclone |
SH30070.03 |
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| L-Glutamine |
Reagent |
Gibco |
25030 |
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| Non-Essential Amino Acids |
Reagent |
Gibco |
11140-050 |
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| Penicllin / Streptomycin |
Reagent |
Gibco |
15140-122 |
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| Sodium Pyruvate |
Reagent |
Gibco |
11360-70 |
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| β-mercaptoethanol |
Reagent |
Chemicon |
ES-007-E |
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| leukemia inhibitory factor(LIF) |
Reagent |
Chemicon |
LIF2005 |
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| 96-well ultralow attachment plate |
Tool |
Corning |
3474 |
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1. Wobus, A. M., Wallukat, G., & Hescheler, J. Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers. Differentiation, 48(3), 173-182. (1991).
2. Metzger, J. M., Lin, W. I., & Samuelson, L. C. Transition in cardiac contractile sensitivity to calcium during the in vitro differentiation of mouse embryonic stem cells. J Cell Biol, 126(3), (1994).
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Wang X, Yang P (2008). In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method. JoVE. 17. http://www.jove.com/index/details.stp?id=825, doi: 10.3791/825
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Allowed tags: i, b, u, sup, sub 
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| | 10/08/2008 7:13:45 AM
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Barbara Tondelli responded with a statement of type: Neutral
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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. |
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| | 10/08/2008 12:09:31 PM
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phillip yang responded with a statement of type: Neutral
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hi,
i work together w/ xiang. he returned to china so i asked him to respond to you but in case he doesn'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 |
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| | 11/07/2008 3:02:11 AM
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Huijun Zhu responded with a statement of type: Neutral
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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, does 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!
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| | 11/10/2008 8:00:57 AM
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Xiang responded with a statement of type: Neutral
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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
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| | 11/18/2008 3:59:48 AM
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Huijun Zhu responded with a statement of type: Neutral
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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 goes wrong?
And how can I purify the cardiaomyocytes? How can I increase the percent of the cardiomyocytes?
Thank you a lot! |
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| | 06/12/2009 10:42:36 AM
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mariateresa responded with a statement of type: Neutral
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Dear Doctor Xiang Wang
is necessary to add gelatin on the plate or not??
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Mailer.URL: http://www.jove.com/index/details.stp?id=825 |
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