A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

A. Microfabrication of the microfluidic device

1. 4-inch Si wafer is treated with reactive oxygen plasma (5 min at 30W, Harrick Scientific, NY).
2. Negative photoresist (SU-8 2015, Microchem, MA) is spin-coated at 900 rpm for 1 min on a Si wafer.
3. The wafer is soft baked at 95°C for 6 min on a hotplate and is exposed to UV light (200W) for 4 min through a mask film containing microchannels.
4. The wafer is post baked at 95°C for 6 min and is developed using SU-8 photoresist developer.
5. The photoresist patterned wafer containing microchannels is placed in a Petri-dish.
6. Poly(dimethylsiloxane) (PDMS) (Sylgard 184) molds are fabricated by mixing silicone elastomer and curing agent (10:1 ratio).
7. The PDMS mixture is poured onto the Si master mold and is placed on a vacuum desiccator to remove bubbles.
8. PDMS is cured at 70°C for 1~2 hours.
9. PDMS molds are then peeled off from the Si master mold.

B. Assembling the device

1. 40 µm thick top fluidic channels and 40 µm thick bottom microgrooved channels (25, 50, 75, and 100 μm wide) are obtained from two different Si master molds.
2. Channel inlet and outlet of the fluidic device are punched.
3. Fluidic channels and microgroove channels are irreversibly bonded by the reactive oxygen plasma (5 min at 30W, Harrick Scientific, NY).
4. Extracellular matrix (ECM) (i.e. fibronectin) is coated inside microfluidic device for 1 hour in incubator (37°C).

C. Cell seeding and experimental setup

1. NIH-3T3 mouse fibroblasts are cultured in a tissue culture flask using Dulbecco's Modified Eagle's Media (DMEM) containing 10% Fetal Bovine Serum (FBS).
2. Cells are trypsinized and dissociated.
3. Dissociated cells are loaded into the microgroove channels at the cell density of 3×10⁶ cells/ml (Figure 1).
   
    
      Figure 1
   
   
4. 2 mL media, 100 mM H₂O₂, and apoptosis assay (20 µL Annexin V and 40 µL propidium iodide, Invitrogen, CA) are infused into a channel using a syringe pump (1 µl/min).
5. Cells are real-time monitored by using an inverted microscope (Nikon TE 2000).

Cells were immobilized and patterned within microgrooved substrates in a microfluidic device. The apoptosis process of cells exposed to hydrogen peroxide was observed in real-time and analyzed by using Annexin V and propidium iodide. Thus, this microfluidic device containing microgroove channels could be useful for high-throughput drug screening.