The overall goal of this procedure is to design and fabricate microfluidic devices for observing how cells respond to environmental stimuli.This method can help answer key questions in the cell biology and biomedical engineering.Such as how cells responses under different environmental stimuli including chemicals, shear stresses, and electric field.The main advantage of this technique is that tubes are easily to be fabricated and assembled.And this these microfludic devices may make the in-vivo condition with high superb capabilities.Demonstrating the procedure will be Tzu-Yuan Chou, an undergraduate student from my lab, and Hsien-San Hou, a post-staff from Dr.Julian Chung's lab.To begin, turn on the carbon dioxide laser scriber and check it's connection to the computer.Then load up a pre-drawn Christmas tree pattern, like one of the patterns shown here into the scriber.Next, place a PMMA sheet or piece of double-sided tape on top of the stage of the scriber.Adjust the focus of the carbon dioxide laser using the calibration bar and the visible helium neon laser.Then scribe the pattern onto the substrate.When finished, pick up the pattern substrate, remove the unwanted pieces, and use a stream of nitrogen to clean the surface of any remaining debris.Repeat this process for each component of the chip.Assemble the chemical shear stress microfluidic chip.To accomplish this, attach three sheets of the chemical shear stress patterned PMMA sheets using two pieces of scribed double-sided tape.Use super glue to attach a acrylic adapters with screw threads in the middle to the top-most PMMA layer of the chip by aligning the screw threads and the holes.These adapters will serve as medium inlets outlets and salt bridge connectors.Then add one more piece of the 07 mm thick double-sided tape to the bottom of the chip, and attach the chip to a 10 cm diameter Petri dish.Once assembled, put the chip in a vacuum chamber for overnight.Now assemble the chemical-electric field chip.Attach the scribed PMMA sheet for the chemical-electric field chip with double-sided tape to a 10 cm diameter Petri dish.Prior to using the chips, expose them to UV for 30 minutes to sterilize them.Next, connect the inlets of the chips to 3 ml PBS-filled syringes via plastic tubes and finger-tight nuts.In the same manner, connect the outlets to a waste tube.Slowly depress the plunger on the syringe to prime the fluidic channels with PBS.Push the syringe back and forth to remove bubbles, then put the chips inside an incubator overnight.To setup the chemical-electric field chips, fill 3%agarous in in the tubular nuts to setup the salt bridges.Then place the silver silver chloride electrodes into the tubular nuts.Grow lung cancer CL1-5 cells in D-MEM supplemented with 10%FBS until they reach 90%confluence.Then aspirate the medium.Wash the cells once with pre-warmed PBS.And add 2 ml of a 05%tripsin buffer solution to a 10 cm diameter plate.Place the cells back at 37
