Advanced Microfluidic Device Designed for Cyclic Compression of Single Adherent Cells

Cells in our body experience different types of stress including compression, tension, and shear. It has been shown that some cells experience permanent plastic deformation after a mechanical tensile load was removed. However, it was unclear whether cells are plastically deformed after repetitive compressive loading and unloading. There have been few tools available to exert cyclic compression at the single cell level. To address technical challenges found in a previous microfluidic compression device, we developed a new single-cell microfluidic compression device that combines an elastomeric membrane block geometry to ensure a flat contact surface and microcontact printing to confine cell spreading within cell trapping chambers. The design of the block geometry inside the compression chamber was optimized by using computational simulations. Additionally, we have implemented step-wise pneumatically controlled cell trapping to allow more compression chambers to be incorporated while minimizing mechanical perturbation on trapped cells. Using breast epithelial MCF10A cells stably expressing a fluorescent actin marker, we successfully demonstrated the new device design by separately trapping single cells in different chambers, confining cell spreading on microcontact printed islands, and applying cyclic planar compression onto single cells. We found that there is no permanent deformation after a 0.5 Hz cyclic compressive load for 6 min was removed. Overall, the development of the single-cell compression microfluidic device opens up new opportunities in mechanobiology and cell mechanics studies.


Device fabrication -SU-8 patterning
Three photomasks were produced by high resolution inkjet printing on transparency film (CAD/Art Services). Four silicon molds were SU-8 patterned by standard photolithography using the three photomasks. The first silicon mold is composed of one layer of SU-8 pattern for PDMS casting of the control layer. The SU-8 pattern of the control layer defined two sets of integrated microfluidic control valves for closing of the main microfluidic channel (trapping control valve) and deflection of the flow layer in the compression chambers (compression control valve) respectively. The second silicon mold is composed of three layers of SU-8 patterns to be used for PDMS spin-coating of the flow layer. The first SU-8 patterning layer defined the side microfluidic pipette channel for trapping cells. The second SU-8 patterning layer defined the compression block for flat planar compression of cells. The third SU-8 patterning layer defined the main microfluidic channel, the trapping chambers, the inlets and the outlet. The third silicon mold is composed of one layer of SU-8 pattern for PDMS casting of the bottom alignment layer.
The SU-8 pattern of the bottom alignment layer has the same design as the third SU-8 patterning layer of the second silicon mold, so that the bottom alignment layer can be used for aligning the fibronectin and the PDMS control/flow substrate. The fourth silicon mold is composed of one layer of SU-8 pattern for PDMS casting of the microcontact printing layer. The microcontact printing layer defines the shape and size for printing fibronectin directly within the compression chambers.
The SU-8 patterning procedures of the four silicon molds followed the standard protocol developed by Microchem, Inc. for SU-8 2000. First, four silicon wafers were dehydrated on a hot plate at 200 o C for 15 min to promote photoresist adhesion. For SU-8 patterning the silicon mold for the control layer, SU-8 2025 was spin-coated at 4000 rpm onto the wafer for 30 s, which gave a thickness of 20 µm. The photoresist was then exposed to UV light for 12 s under a contact aligner (Karl Suss, MJB45). For the first SU-8 patterning of the flow layer, SU-8 2005 was spin-coated at 2700 rpm onto the silicon wafer for 30 s, which gave a thickness of 5.4 µm.
The photoresist was then exposed to UV light for 9 s with the first flow channel photomask, which defined the side pipette arrays. After development of first SU-8 layer, the silicon wafer was hard-baked at 200 o C for 20-30 minutes to further cross-link the developed SU-8 patterns before the application of the second SU-8 layer. Subsequently, for the second-layer SU-8 patterning of the flow layer, SU-8 2025 was spin-coated at 3500 rpm onto the silicon wafer for 30 s, which gave a combined thickness of 24 µm. The photoresist was then aligned with the second flow channel photomask, which defined the compression block, and exposed to UV light Supplemental Materials for 11.5 s. After development of SU-8, both silicon molds were hard-baked at 200 o C for 20-30 minutes to further cross-link the developed SU-8 patterns before the application of the third SU-8 layer. Subsequently, for the third-layer SU-8 patterning of the flow layer, SU-8 2025 was spincoated at 2100 rpm onto the silicon wafer for 30 s, which gave a combined thickness of 38 µm.
The photoresist was then aligned with the third flow channel photomask, which defined the main microfluidic channel, and exposed to UV light for 12.5 s. For SU-8 patterning the silicon mold for the bottom alignment layer, SU-8 2010 was spin-coated at 2000 rpm onto the wafer for 30 s, which gave a thickness of 13 µm. The photoresist was then exposed to UV light for 9 s under a contact aligner. For SU-8 patterning the silicon mold for the microcontact printing layer, SU-8 2025 was spin-coated at 4000 rpm onto the wafer for 30 s, which gave a thickness of 20 µm.
The photoresist was then exposed to UV light for 12 s under a contact aligner. After