Preparation of cell cultures:

1. Start with silicon parts coated with plasma-treated polystyrene.
2. Coat parts with appropriate extracellular matrix proteins to support attachment of desired cell type. For hepatocytes, incubate in 50 g/ml Collagen-1 solution at 37°C for 45 min. For 3T3 fibroblasts, no matrix is needed.
3. Lock parts with complementary parts in contact configuration.
4. Soak in 70% ethanol for a minimum of 10 min to sterilize. Rinse 2x in ddH₂O, and 1x in cell culture media.
5. Seed cells at a concentration of 500,000 cells/ml in the appropriate culture medium. Use 1 ml in each well of a 12-well culture plate.
6. Incubate for 1 hr, shaking every 15 min to re-suspend cells evenly.
7. If a confluent monolayer has not been achieved after 1 hr, aspirate the cell suspension and repeat seeding with a fresh suspension. Repeat until the desired cell density has been achieved.
8. Remove the complementary parts. Transfer parts to fresh wells and incubate overnight to allow cells to adhere and spread fully.
9. Form co-cultures by locking appropriate parts into either the gap or contact configuration. The configuration may be changed at any desired point during culture.
10. When changing media, take care to leave cells dry for as little time as possible, preferably just a second or two. Draw out fluid with one hand and immediately replace media using the other hand.

Processing silicon parts for reuse:

1. Strip cells by soaking in bleach and rinsing with water.
2. Allow parts to dry completely. Soak in toluene for 2 h to strip polystyrene.
3. Clean in piranha solution (1:2 H₂O₂:H₂SO₄) heated to 120°C.
4. Rinse for 15 min under a continuous flow of ddH₂O. If you are not going to deposit polystyrene immediately, store the parts in water.
5. Dissolve polystyrene in toluene at 100 mg/ml. Vortex in polypropylene conical for about 1 hr, or until fully dissolved. A little more than 1 ml of solution is required for every 10 parts.
6. Spin coat polystyrene solution on individual silicon parts at 2,400 rpm for 30 sec.
7. Bake for at least 5 h at 120°C.
8. Treat with oxygen plasma (200 mTorr, 200 W) for 1 min.