Name: Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies (Video) | JoVE Journal
Uploaded: 2008-03-05T22:18:00.000+00:00
Duration: 7 min 23 s
Description: Learn a simple method for embryoid body formation using microfabricated wells with controlled cell numbers. This technique reduces manual labor compared to traditional hanging drop methods.

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5 minutes

We would like to thank CIRM for support of this work. The cell line was generously donated from Dr. Andras Nagy at Mount Sinai Hospital, Toronto, Ontario.

1. Making Shrinky-Dink Mold
<ol>
<li>Print the desired pattern on shrinky-dink sheet using a good definition printer.</li>
<li>Bake shrinky-dink sheet at 163° C for about 10 minutes, or until fully shrunk and having aquired a regular shape.</li>
<li>After shrinky-dink mold has cooled down, submerge it in an isopropanol bath until the complete surface is barely covered.</li>
<li>Carefully, spray some acetone over the mold and shake container a few times. Add more isopropanol to wash out acetone excess and repeat this step a few times until shrinky-mold looks clean.</li>
<li>Immerse mold in distilled water for 10 minutes to wash off any remaining organic solvent.</li>
<li>Air clean shrinky-mold. Re-heat it for about 5 minutes at 163° C. This will compact ink and evaporate any remaining solvent.</li>
</ol>
2. Making PDMS microwells
<ol>
<li>Prepare a 10:1 PDMS/curing agent mixture, and agitate vigorously for few minutes.</li>
<li>Place shrinky-dink mold in a small petri dish. Pour PDMS mixture until it reaches about 1/2 cm over the mold surface.</li>
<li>Place dish under vacuum bell to eliminate all bubbles from PDMS mixture.</li>
<li>Place dish in oven at 70°C, overnight.</li>
<li>Cut off solid PDMS from mold and bind it to a glass slide just by applying pressure.</li>
<li>Discard first microwell-chip, since it has ink residues incrusted between PDMS.</li>
<li>Repeat this procedure to produce a second chip that is ink-free and has a more defined shape.</li>
<li>Clean microwell-chip using 70% ethanol solution. Place it under UV light source for 10 minutes to sterilize it.</li>
</ol>
3. Trapping cells in microwells
<ol>
<li>Count cells and dilute them in culture media to desired concentration (depending on how many initial cells in wells you would like). For example, to get approximately 10-15 cells per well (average = 11, SD = 5.4, loading rate= 93%), we used a concentration of 8×104 cells/ml. For a concentration of 17×104 cells/ml, we could reliably get between 25 and 35 cells per well (average = 27.17857 SD = 7.7, loading rate = 100%).</li>
<li>Carefully place microwell chip in a 50 ml centrifuge tube containing a solidified PDMS base.</li>
<li>Add about 2JDP ml of the cell solution.</li>
<li>Centrifuge for 5 minutes at 760 rpm and 4°C.</li>
<li>Pipet out excess solution and carefully wash microwell with PBS 1X solution.</li>
<li>Place microwell in a small petry dish, being careful while taking the chip out of the centrifuge tube.</li>
<li>Wash cell excess using 1 X PBS solution.</li>
<li>Place microwell chip under an inverted microscope to verify intended number of cells per well.</li>
<li>Incubate microwell containing cells at standard conditions. <img src="/files/ftp_upload/old/692_2008_7_21_1/692_Figure-01.png" alt="Microscopy diagram of cells; cellular distribution and structure analysis." width="350" height="183" data-altupdatedat="1787630408000" data-alt="Figure-01"></li>
</ol>
4. Cell incubation  
<ol>
<li>Follow the normal EB protocol in the lab.</li>
<li>Change the medium slowly from the side of the chamber; avoid disturbing the cell in the microwell.</li>
</ol>

<ol><li>Keller, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+differentiation+of+embryonic+stem+cells%5BTitle%5D)+AND+%22Curr+Opin+Cell+Biol%22%5BJournal%5D)">In vitro differentiation of embryonic stem cells.</a> Curr Opin Cell Biol. 7, 862-869 (1995).</li>
<li>Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W., Kemler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+in+vitro+development+of+blastocyst-derived+embryonic+stem+cell+lines%3A+Formation+of+visceral+yolk+sac%2C+blood+islands+and+myocardium%5BTitle%5D)+AND+%22Journal+of+Embryology+and+Experimental+Morphology%22%5BJournal%5D)">The in vitro development of blastocyst-derived embryonic stem cell lines: Formation of visceral yolk sac, blood islands and myocardium.</a> Journal of Embryology and Experimental Morphology. 87, 27-45 (1985).</li>
<li>Park, J., Cho, C. H., Parashurama, N., Li, Y., Berthiaume, F., Toner, M., Tilles, A. W., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfabrication-based+modulation+of+embryonic+stem+cell+differentiation%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Microfabrication-based modulation of embryonic stem cell differentiation.</a> Lab Chip. 7, 1018-1028 (2007).</li>
<li>Koike, M., Sakaki, S., Amano, Y., Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+embryoid+bodies+of+mouse+embryonic+stem+cells+formed+under+various+culture+conditions+and+estimation+of+differentiation+status+of+such+bodies%5BTitle%5D)+AND+%22J+Biosci+Bioeng%22%5BJournal%5D)">Characterization of embryoid bodies of mouse embryonic stem cells formed under various culture conditions and estimation of differentiation status of such bodies.</a> J Biosci Bioeng. 104, 294-299 (2007).</li>
<li>Hwang, N. S., Varghese, S., Elisseeff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Controlled+differentiation+of+stem+cells%5BTitle%5D)+AND+%22Advanced+Drug+Delivery+Reviews%22%5BJournal%5D)">Controlled differentiation of stem cells.</a> Advanced Drug Delivery Reviews. 60, 199-204 (2008).</li>
<li>Adelman, C. A., Chattopadhyay, S., Bieker, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+BMP%2FBMPR%2FSmad+pathway+directs+expression+of+the+erythroid-specific+EKLF+and+GATA1+transcription+factors+during+embryoid+body+differentiation+in+serum-free+media%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The BMP/BMPR/Smad pathway directs expression of the erythroid-specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum-free media.</a> Development. 129, 539-549 (2002).</li>
<li>Tanaka, N., Takeuchi, T., Neri, O. V., Sills, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laser-assisted+blastocyst+dissection+and+subsequent+cultivation+of+embryonic+stem+cells+in+a+serum%2Fcell+free+culture+system%3A+applications+and+preliminary+results+in+a+murine+model%5BTitle%5D)+AND+%22J+Transl+Med%22%5BJournal%5D)">Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model.</a> J Transl Med. 4, (2006).</li>
</ol>

We have developed a simple, rapid, and scalable culture method to load pre-defined numbers of cells into microfabricated wells (molded from Shrinky-Dinks) and maintain them for embryoid body development. Finally, these cells are easily accessible for further analysis and experimentation. This method is amenable to any lab and requires no dedicated equipment because we obviate the need for photolithography. We can vary the size of the microwells as well as the concentration of cells/ wells to change the number and size of...

<table><tbody><tr><td></td><td></td><td></td><td></td></tr><tr><td>Material Name</td><td>Type</td><td>Company</td><td>Catalogue Number</td></tr><tr><td>Shrink-Dink Film</td><td>Material</td><td>K&amp;amp;B Innovations</td><td>D300-10A</td></tr><tr><td>PDMS</td><td>Material</td><td>Dow Corning</td><td>Sylgard 184</td></tr><tr><td>Acetone</td><td>Reagent</td><td>Fisher Scientific</td><td>A16P-4</td></tr><tr><td>Ethanol</td><td>Reagent</td><td>Fisher Scientific</td><td>A405P-4</td></tr><tr><td>PBS</td><td>Reagent</td><td>Sigma</td><td>P4417</td></tr><tr><td>BMP-4</td><td>Reagent</td><td>R and D systems</td><td>314-BP-010</td></tr><tr><td>Knock-Out DMEM (KO DMEM)</td><td>Reagent</td><td>Invitrogen</td><td>10829</td></tr><tr><td>KnockOut Sirum Replacement (KSR)</td><td>Reagent</td><td>Invitrogen</td><td>10828</td></tr><tr><td>Penn-Strep</td><td>Reagent</td><td>Invitrogen</td><td>15070-063</td></tr><tr><td>L-glutamine</td><td>Reagent</td><td>Invitrogen</td><td>25030-081</td></tr><tr><td>Non-essential Amino Acids (NEAA)</td><td>Reagent</td><td>Invitrogen</td><td>11140</td></tr><tr><td>D-mercaptoethanol (BME)</td><td>Reagent</td><td>Calbiochem</td><td>444203</td></tr><tr><td>Leukemia Inhibitory Factor (LIF) {ESGRO 106 units}</td><td>Reagent</td><td>Chemicon</td><td>ESG1106</td></tr><tr><td>Printer</td><td>Tool</td><td>HP</td><td>Laser Jet 2420d</td></tr><tr><td>Oven</td><td>Tool</td><td>Yamato Scientific</td><td>DP-22</td></tr><tr><td> For the mESC media(McCloskey lab protocol): (for total media prepared: 50ml; 100ml) KO DMEM: 40.8ml; 81.6ml 15% KSR: 7.5ml; 15ml 1x Penn-Strep: .5ml; 1ml 2mM L-glutamine: .5ml; 1ml NEAA : .5ml; 1ml LIF: 100ul; 200ul BMP-4 (10ng/ml): 50ul; 100ul Diluted BME: 50ul; 100ul (Add 35ul of sterile filtered BME to 5ml of PBS and syringe filter sterilize. Discard after 2 weeks. Final concentration in the solution is .1mM) </td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr></tbody></table>

shrinky-dink hanging drops: a simple way to form and culture embryoid bodies

Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies

Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious approach of pipetting an arbitrary number of cells into well plates. The interactions between the stem cells forced into close proximity of one another promotes the generation of the EBs. Because the media in each of the wells has to be manually exchanged every day, this approach is manually intensive.
Moreover, because environmental parameters including cell-cell, cell-soluble factor interactions, pH, and oxygen availability can be functions of EB size, cell populations obtained from traditional hanging drops can vary dramatically even when cultured under identical conditions. Recent studies have indeed shown that the initial number of cells forming the aggregate can have significant effects on stem cell differentiation. We have developed a simple, rapid, and scalable culture method to load pre-defined numbers of cells into microfabricated wells and maintain them for embryoid body development. Finally, these cells are easily accessible for further analysis and experimentation. This method is amenable to any lab and requires no dedicated equipment. We demonstrate this method by creating embryoid bodies using a red fluorescent mouse cell line (129S6B6-F1).

We show a simple and rapid method to load pre-defined numbers of cells into microfabricated wells and maintain them for embryoid body development.

Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious ...

Embroid bodies are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method. A laborious approach of pipetting, an arbitrary number of cells into well plates.The process is manually intensive because the media and each well plate has to be manually exchanged each day. Moreover, though, recent studies have shown that the initial number of cells forming the aggregate can have effects on stem cell differentiation. There is no way of predetermining the number of cells using this method.In this video, we show a way to simply and rapidly load different numbers of cells into micro fabricated wells for embroid body development. Hi, I'm Jonathan Pagan from the lab of Michelle K in the School of Engineering at the University of California Merced, And I'm a Jesus Luna also from the Ks lab. Today we'll show you a procedure for creating embroid bodies and micro fabricated wells by centrifuge.This procedure involves the following steps, creating a micro device, Loading the stem cells And feeding the stem cells. So let's get started. The first step in this procedure is to make the shrinky dink mold using a good definition printer.Print the desired pattern on shrinky dink sheets. Next, bake the shrinky D sheet at 160 degrees Celsius for about 10 minutes or until it has fully shrunk and acquired a regular shape. After the shrinking ink mold has cooled down for two minutes and is cooled to the touch.Submerge it in an isopropanol bath until the complete surface is barely covered. After the shrinking ink mold has been submerged in the isopropanol, carefully spray some acetone over the mold and shake the container a few times. Then add more isopropanol to wash out acetone excess, and repeat this step a few times until the shrinking ink mold looks clean.Next, immerse the mold and distilled water for 10 minutes to wash off any remaining organic solvents. After our immersion in water, air cleaned the shrinky ink mold using compressed air and reheat it for about five minutes at 160 degrees Celsius. This will compact the ink and evaporate any remaining solvent.And now we can proceed to making the PDMS microwells Now. Now we Generate the PDMS microwells, which will be used to culture the stem cells for embroid body development. Prepare a 10 to one PDMS to curing agent mixture and agitate vigorously for a few minutes.Next, pour the PDMS mixture over the shrinky dink mold in a small Petri dish until it reaches about one half centimeter over the mold surface. After pouring on the PDMS bubbles can often be observed over the mold to remove them. Place the dish under a vacuum bell for 20 minutes to eliminate all bubbles from PDMS mixture.After ensuring that all bubbles have been removed, place the dish in the oven at seven degrees Celsius for 20 minutes. After the incubation, cut off solid PDMS from the mold With a razor blade. Clean it using tape and bind it to A glass slide with the wells facing up.This is done simply by applying pressure to the edges. Next, clean it using 70%ethanol solution and place it under a UV light source for 10 minutes. To sterilize it.The first time you do this, you're gonna discard the PDMS. This will help take off any extra ink residue from your mold. You can use the mold as is thereafter.Repeat the molding procedure once more to produce a second chip that is ink free and has a more defined shape. Make sure to current a discharge for 20 seconds before starting to create a hydrophilic surface for easy loading. After making the PMS Microwells, they can now be used for the study of Enid bodies.So let's proceed. To begin, cells must be counted and diluted in cell culture media to desired concentration, which depends on how many initial cells in wells you would like. At 1.5 times 10 to the fourth cells per milliliter, we found that you get 14 cells per well with every well loaded.Add two milliliters of cell solution to a 50 milliliter centrifuge tube containing a solidified PDMS base. Carefully place the micro well chip into the centrifuge tube centrifuge for five minutes at 760 RPM. Next pipette out media and place the micro well in a small Petri dish.Be careful not to spill while taking the chip out of the centrifuge tube. Next place the micro well chip under an inverted microscope to verify that every well is loaded. Place the chip into the holder and incubate the micro wells containing cells at standard conditions for 24 hours.In order to maintain the cells, the medium can be changed slowly from the side of the chamber after 24 hours. Avoid disturbing the cells in the micro. Well, you can do this every day.The nice thing is that you pipette once and exchange all the media as opposed to having to exchange the media for each well, for conventional hanging drops, We've just shown You how to culture cells and create embroid bodies from shrinky dink molds. This method is extremely useful because it's simple, rapid, and manually, less intensive than the hang drop method. This method requires no photolithography.Plus we can control the number of cells we start our embroid bodies from. So that's it. Thanks for watching and good luck with your experiments.

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Learn a simple method for embryoid body formation using microfabricated wells with controlled cell numbers. This technique reduces manual labor compared to traditional hanging drop methods.

Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies

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