A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

This pneumatically activated microfluidic compression device can be used to study mechanobiology in 3D hydrogel cultures of growth plate chondrocytes. The microfluidic device is cost and time-effective because it can simultaneously generate multiple magnitudes of a compression on small volumes of samples, and various microscopic imagings can be applied with the device. Our method can be also applied to the study of mechanobiology of other cell types.

To set up the microchannel layer, first mix PDMS with a weight ratio of 10 prepolymers to one curing agent for five minutes. Next place the mold on a foam pad and pour the mixture into the master mold. Degas the PDMS in a vacuum chamber for 30 minutes and sandwich the degassed PDMS with a piece of transparency film.

Clamp the sandwiched assembly with the glass plate, foam pads, and plexiglass plates. Cure the PDMS at 80 degrees Celsius for six hours and isolate the PDMS layer and the transparency film from the sandwiched structure. Activate the surfaces of the PDMS and a clean glass plate with the plasma cleaner for one minute before bonding the PDMS onto the glass plate in the 80-degree Celsius oven for 30 minutes.

Then remove the transparency film. For preparation of the thin PDMS membrane, spin coat uncured PDMS on a transparency film at 1, 000 rotations per minute for one minute to obtain a 60 micrometer thick PDMS layer. Partially cure the spin coated PDMS at 80 degrees Celsius for 20 to 30 minutes before using the plasma cleaner to activate the microchannel PDMS layer on the glass plate and the thin PDMS layer for one minute.

Then bond the thin PDMS membrane layer onto the microchannel PDMS layer and place the layers in the 80-degree Celsius oven overnight. For tubing block preparation, place metal tubes vertically on a petri dish and gently pour uncured PDMS into the dish until about 3/4 of the tubes are submerged. After degassing the PDMS in a vacuum chamber for 30 minutes, cure the PDMS in the oven at 60 degrees Celsius for more than six hours.

At the end of the curing process, cut a piece of PDMS block containing one tube and punch a hole in the thin PDMS layer for the inlet. Activate the PDMS block and the thin PDMS layer with the plasma cleaner. After one minute, attach the PDMS block to the inlet hole of the thin PDMS layer and place the entire actuation unit in the oven at 80 degrees Celsius overnight.

For agarose gel mold preparation, mix 5%agarose and 200 millimolar calcium chloride in deionized water and heat the agarose gel solution until boiling. Pour the boiled agarose gel solution into an aluminum mold and sandwich the mold with the glass plate. After five minutes, remove the solidified agarose gel from the aluminum mold.

Plate 150 microliters of alginate-chondrocyte solution onto the aminosilanized glass plate and cover the solution with agarose gel mold for three minutes. At the end of the incubation, use a razor blade to remove the excessive alginate gel solution and remove the agarose gel mold from the plate. Then place the alginate-chondrocyte constructs into cross-linking solution containing 50 millimolar calcium chloride and 140 millimolar sodium chloride in deionized water for one minute.

To assemble the device, locate four one-millimeter thick PDMS spacers onto the four corners of the thin PDMS layer of the actuation unit. Cover the air chambers of the thin PDMS layer with 700 microliters of chondrocyte culture medium. Using a stereo microsope, place the alginate-chondrocyte construct on the thin PDMS layer, taking care to align the constructs with the air chambers, and clamp the device with 3D-printed clamps.

For actuation of the device, use a piece of silicon tubing to connect the outlet of an air pump with the inlet of a solenoid valve. Use an additional piece of tubing to connect the outlet of the solenoid valve with the inlet of the assembled device. Connect the solenoid valve with a function generator and use a one hertz square wave generated by the function generator to manipulate the solenoid valve.

Then turn on the air pump to actuate the device pneumatically. The microfluidic chondrocyte compression device contains five by five arrays of cylindrical alginate-chondrocyte constructs and these constructs can be compressed with five different magnitudes of compression. In this example, the gel column was compressed by 33.8%in height by the largest PDMS balloon and the resultant compressive strain of the gel constructs increased by approximately 5%per 0.2 millimeter increment and the PDMS balloon diameter.

Compressive deformation of the chondrocytes was determined by imaging the cells in a 613 by 613 by 40 to 55 micrometer volume near the gel construct center. Here an image of a chondrocyte that was compressed by 16%by the largest PDMS balloon is shown. In this graph, the distribution of the measured cell compression strain values can be observed.

In this analysis, the cells were compressed more overall by larger PDMS balloons. Taken together, these data suggest that the amounts of alginate gel and chondrocyte compression are controlled by the diameter of the PDMS balloons under a constant pressure of 14 kilopascals. To ensure the repeatability of the device performance, creating a thin PDMS membrane with a constant thickness and elasticity is crucial.

Following this procedure, various biological assays, such as immunofluorescence and in situ hybridization can be performed. Using this technique, many questions about mechanotransduction in growth plate or articular chondrocytes can be answered.