1. Making Shrinky-Dink Mold

1. Print the desired pattern on shrinky-dink sheet using a good definition printer.
2. Bake shrinky-dink sheet at 163° C for about 10 minutes, or until fully shrunk and having aquired a regular shape.
3. After shrinky-dink mold has cooled down, submerge it in an isopropanol bath until the complete surface is barely covered.
4. 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.
5. Immerse mold in distilled water for 10 minutes to wash off any remaining organic solvent.
6. Air clean shrinky-mold. Re-heat it for about 5 minutes at 163° C. This will compact ink and evaporate any remaining solvent.

2. Making PDMS microwells

1. Prepare a 10:1 PDMS/curing agent mixture, and agitate vigorously for few minutes.
2. Place shrinky-dink mold in a small petri dish. Pour PDMS mixture until it reaches about 1/2 cm over the mold surface.
3. Place dish under vacuum bell to eliminate all bubbles from PDMS mixture.
4. Place dish in oven at 70°C, overnight.
5. Cut off solid PDMS from mold and bind it to a glass slide just by applying pressure.
6. Discard first microwell-chip, since it has ink residues incrusted between PDMS.
7. Repeat this procedure to produce a second chip that is ink-free and has a more defined shape.
8. Clean microwell-chip using 70% ethanol solution. Place it under UV light source for 10 minutes to sterilize it.

3. Trapping cells in microwells

1. 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%).
2. Carefully place microwell chip in a 50 ml centrifuge tube containing a solidified PDMS base.
3. Add about 2JDP ml of the cell solution.
4. Centrifuge for 5 minutes at 760 rpm and 4°C.
5. Pipet out excess solution and carefully wash microwell with PBS 1X solution.
6. Place microwell in a small petry dish, being careful while taking the chip out of the centrifuge tube.
7. Wash cell excess using 1 X PBS solution.
8. Place microwell chip under an inverted microscope to verify intended number of cells per well.
9. Incubate microwell containing cells at standard conditions.
   
   

4. Cell incubation  

1. Follow the normal EB protocol in the lab.
2. Change the medium slowly from the side of the chamber; avoid disturbing the cell in the microwell.