A second layer of photoresist (SU-8 2075), which defined the overall culture chamber structure and walls between each compartment, was next spun onto the wafer. This layer was then soft baked for 10 minutes at 65°C and 50 minutes at 95°C, and then allowed to cool to room temperature. On top of this SU-8 2075 layer, another layer of SU-8 2025 was spun at 1,000 rpm for additional chamber height. Together, the chamber height was estimated to be 320 μm. The wafer was then aligned to the first layer, such that microchannels spanned the gap between side chambers and central chamber. Next, the wafer was exposed to UV and developed in SU-8 developer. A slow hard-baking protocol was followed by ramping the temperature from room temperature (20°C) to 105°C, which was held for 20 minutes, and then ramped down back to room temperature. This step is important to reduce pealing of SU-8 layers from the wafer due to residual stress.
