Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This is a novel, lithography-free protocol for modeling a 3D skin-on-chip using micromachined, adhesive vinyl layers and a parallel-flow methodology for the dermal compartment generation. Micromachined vinyl provides more simplicity in the fabrication process and versatility in the layout of the device, overcoming some of the limitations of PDMS and traditional lithography approaches. This multilayered skin-on-a-chip can be used for drug and cosmetic testing approaches, since it allows high-throughput screening at lower costs and could be used to model several diseases.

Begin by designing a microfluidic chip using Brother Canvas Workspace software. Create a 30-by-30 centimeter workspace, and fill it with the design patterns for the different layers of the chip before storing it in a svg file. Cut the 30-by-30 centimeter vinyl sheets.

Stick it to a low-tack adhesive mat and eliminate all air bubbles, as necessary. Using a USB drive, upload the svg file to the edge plotter, and set the cutting parameters as cutting pressure at level zero, cutting blade at level three, and level one for the cutting speed. When parameters are set, place the adhesive mat with the vinyl into the plotter and start the process of cutting the channel patterns on 95-micrometer-thick vinyl.

Assemble the whole device by using an aligner for proper adjustment channels, inlets, and outlets. Pile up for vinyl layers with a corresponding micromachine design for assembling the lower channel, keeping the cover tape of the bottom layer to avoid sticking to the aligner. Cut and place the polycarbonate porous membrane on top of the lower channel without covering the inlets.

Add 10 vinyl layers with the upper-chamber design and stick a double-sided tape final layer on top. Remove the chip from the aligner to stick it on the glass slide. Place a two-millimeter-thick PDMS sheet on top of the double-sided tape vinyl layer.

To ensure that the chip is completely watertight, leave a weight on top of the chip overnight. On the next day, sterilize the chip by flushing it with 70%ethanol for five minutes, followed by a wash with distilled water. Connect pump one and pump two to the upper chamber inlet one and the upper chamber inlet two, respectively.

Connect pump three to the lower chamber inlet, then connect the upper chamber and lower chamber outlets to a waste tub. Connect the syringes to each inlet using PTFE tubes and 18-gauge stainless steel connectors. Pump PBS with pump three through the lower chamber inlet at 50 microliters per minute, and with pump two through the upper chamber inlet two at 100 microliters per minute.

Then, load the syringe with the fibrinogen pre-gel, immediately place it into pump one, and run it at 200 microliters per minute. Once the pre-gel exits the upper chamber outlet, stop pumps one and two, then, without removing the tubing, leave the chip to allow gellation at 37 degrees Celsius for 10 minutes. After gellation, block the upper chamber inlet one with a cap, and pump the culture medium through the upper chamber inlet two with pump three at 50 microliters per hour overnight.

24 hours after the generation of the dermal compartment, check that human primary fibroblast cells are spread in the assembly, then introduce five times 10 to the 6 HKCs per milliliter through the upper channel inlet two at 40 microliters per minute for one minute. For cell attachment, place the chip overnight at 37 degrees Celsius in a humidity-saturated incubator. Start pumping fresh culture medium with pump three only through the lower chamber inlet at 50 microliters per minute.

In the representative analysis, the height of the hydrogel along the upper chamber of the assembly is displayed. An average height of 550 micrometers was optimum for the functioning of the gel at the flow rates of 100 and 200 microliters per minute for the sacrificial PBS and the pre-gel, respectively. Failure to flush PBS through the lower channel led to discrepancies between the theoretical height of the gel and the one measured, with a difference of 40%After 24 hours from initiation of the parallel-flow protocol, the human primary fibroblasts were found to be successfully spread in the fibrin gel in the upper chamber of the assembly, following which, green fluorescent protein-expressing HKCs could be effectively seeded on top of the hydrogel.

Removal of tubing without keeping the system closed overnight to allow the HKCS to sediment resulted in a non-uniform, confluent monolayer of cells. 3D confocal analysis of the undifferentiated skin model in the microfluidic chip clearly showed the surface of the hydrogel separating HKCs in the top from the fibroblast embedded at the bottom. The generated skin could be exposed to the air/liquid interface to obtain a mature and differentiated epidermis, and further be characterized using several dermal and epidermal markers.

This technique allows for a faster and cheaper device fabrication, and the generation of multilayered skin using microfluidics, providing a more realistic approach for drugs and cosmetics testing.