We're developing modular microphysiological systems that mimic human barrier tissues. Our system is unique because it can be reconfigured from a static open-well culture to a flow-enhanced system. This flexibility allows the platform to be used in both bioscience and engineering focused laboratories.
We present a protocol for creating a reconfigurable open-well platform with flow-enhanced culture and lifestyle imaging. It is compatible with conventional bioscience protocols. Our reconfigurable design allows users to switch between the open-well and microfluidic modes during an experiment and allows the user to conduct each step using a format they are comfortable with.
Begin by fabricating the patterning stencil for the core module well. To do so, laser cut the acrylic sheet according to the desired design to form an array of cavities as displayed on the screen. Then remove the protective layer from the pressure sensitive adhesive or PSA film fixed to the sheet.
Attach the acrylic sheet to the mold. Next, maintaining a 10 is to 1 base to curing agent ratio thoroughly mix the polydimethylsiloxane or PDMS prepolymer. Degas the mixture in a vacuum chamber until visible bubbles are removed.
Slowly pour the degas PDMS into the mold cavities while leveling it with a flat edge. Cure the PDMS in the mold on a hot plate at 70 degrees Celsius for one hour. Allow it to cool to room temperature.
Then using flat tip tweezers, extract the stencils from the mold cavities. To fabricate the flow module, laser cut the acrylic sheet to form an array of clover-shaped cavities as shown on the screen. Remove the protective layer from the PSA film fixed to the laser cut sheet, and attach the sheet to the silicon mold by aligning the triangular alignment marks.
Slowly pour the degas PDMS into the mold cavities and level it with a flat edge. Place the mold on a hot plate and bake the PDMS at 70 degrees Celsius for one hour. Allow the mold to cool to room temperature.
Using flat tip tweezers, carefully remove the flow modules from the mold cavities. Orient the flow module so that the microchannel features face upward. With the help of a biopsy punch, create the core inlet and outlet ports at the end of the channel.
For fabricating the lower and upper acrylic housing, laser cut a two millimeter acrylic sheet to create the upper housing with holes for magnet insertion. Similarly, laser cut the acrylic sheet to generate the lower housing with the magnetic insertion holes. After removing the PSA protective layer from the sheets, attach the lower housing to a glass cover slip.
Pressed to fit 4.75 millimeter nickel plated neodymium magnets into the laser cut holes of the housings. The open-well core module fitted within the specific cavity created by the lower housing and the cover slip. Subsequently, the flow module, including the microchannel and access ports fitted into the well of the core module.
The flow module was securely sealed against the silicon support layer of the membrane by the magnets embedded in the lower and upper housings. The removable stencil was designed to fit within the open-well of the core module and provided a specific window for cells to settle preferentially on the membrane surface. To begin, coat the membrane of the assembled reconfigurable cell culture platform with five micrograms per square centimeter of fibronectin for one hour at room temperature to improve cell attachment.
Then using a P200 pipette, aspirate the coating solution. Place the patterning stencil in the core module well. Add cell media to the well and bottom channel of the device.
Seed human umbilical vein endothelial cells into the well. Place the device in a petri dish. Add a 15 milliliter conical tube cap containing deionized water to the petri dish to maintain local humidity.
Transfer the petri dish setup to the incubator. To culture the cells under flow, proceed to reconfigure the platform from open-well to the microfluidic mode. To do so, fill the reservoirs and tubes with endothelial cell growth media.
Place the lower housing on the sample stage. Insert the open-well core module into the lower housing. Then remove the stencil, aspirate the cell media from the well of the module, and place the flow module in the well.
Next, place the upper housing to seal the flow module in the core module well. Connect the inlet and outlet dispensing tips to the flow module ports. Start the peristaltic pump to introduce fluid flow into the system.
Stop the pump when the desired time for sheer flow exposure is reached. Remove the inlet and outlet dispensing tips. Then remove the upper housing.
Gently remove the flow module from the well using tweezers. Finally, add 100 microliters of cell media to the well before conducting the desired assay. The cells cultured under flow aligned along the flow direction, while cells cultured without flow remained randomly oriented.
The exposure of cells to sheer stress resulted in the upregulation of kruppel-like factor 2 and endothelial nitric oxide synthase, which serve critical roles and healthy blood vessels.
