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.
