MOLDING OF THE CHIP
1. Fill the graduated plastic cup with 12 mL of the 4Dcell PDMS elastomer.
2. Pipet 1mL of 4Dcell curing agent in the graduated plastic cup. You should have 13 mL in the container.
3. Homogenize the mix for 2 minutes using the glass spatula.
NB: You should see bubbles forming in the mixture!
4. Remove trapped bubbles by degassing the mix with the desiccator for about 10 minutes. You can also let the plastic cup at room temperature and wait at least one hour.
5.Pour the PDMS into the epoxy mold.
6. Degas once again until there is no bubbles left.
7. Place the mold in the oven at 80°C for 1 hour. If you don’t have any oven, you can still let the mold at room temperature and wait for about 8 hours.
8. Once cooled down, gently unmold the chip in alignment with the microchannels. N.B: Do not touch the chip with your fingers (even with the gloves) on the microchannels side, oil will cause unattachment of the chip against the fluorodish.
9. Take the white adhesive tape and use it as a support. First, take off any dust on the PDMS chip. Then, using the sharpened punches round, cut two Ø3 mm holes at the entry and exit of the channels.
10. Place your PDMS chip (the microchannels side on top) and the fluorodish in the plasma cleaner. Activate them for 2 minutes.
11. Directly after activation, bond the PDMS device at the center of the bottom of the fluorodish and press it softly, avoiding pressure on the microchannels area. Put it in the oven for 15 minutes at 80°C to help the bonding of the chip.
PLATING THE CELLS
1. Fill the entry of the microchannels with 20 µL of the surface coating solution, fibronectin. Incubate 30 minutes. N.B: The solution should infiltrate by capillary forces into the microchannels after simply placing a drop of solution at one of the ports.
2. After coating the fibronectin, extensively wash the device with PBS. Load with culture medium (3mL) and incubate the device at a temperature of 37 °C for 15 min. N.B: The PDMS absorbs molecules in the medium. So if you have fragile cells, an overnight incubation with PBS (3mL) at 37°C is recommanded before seeding the cells.
3. Remove the medium, and place a 20 µL droplet of cell solution at one inlet of the device and tilt it to introduce the cells into the microchannels. Incubate 20 minutes (37°C, 5% CO2) to allow cell adherence.
N.B: We advise a cell concentration ranging from 3 to 4.106 cells/mM for classical cell line solution. Otherwise, concentration should be adjusted to have a confluency of 60-70% inside the hole.
4. Finally, add 3mL of culture medium in the fluorodish, close the lid, and incubate it into an appropriate incubator (classically 37°C, 5% CO2 for mammalian cells).
N.B: Incubate overnight before observing cells into the channels.