Here we present protocols for culturing bone cells within an adaptable lab-on-a-chip platform. These techniques can be used to further our understanding of the mechanisms that regulate mechanically-induced bone remodeling. Our system allows for long-term culturing of bone cells, which enables direct quantification of bone formation and resorption in response to altered mechanical loading environments.
Use of these foundational techniques can lead to discoveries that extend to a number of bone-related issues such as osteoporosis, fracture healing, and periprosthetic osteolysis. To create the well and microchannel layer, combine the PDMS prepolymer and curing agent at a ratio of 10 to one in a plastic cup and mix vigorously, then degas the polymer for 30 minutes. Slowly pour the mixture over the appropriate pre-leveled mask.
Let it sit for 30 minutes and bake it at 45 degrees Celsius for 18 hours. Loosen the edges of the PDMS with a tapered laboratory spatula and carefully peel it away from the mask, then cut out individual chips with a scalpel and a 3D-printed template and surface clean them with packaging tape. To make the PDMS membrane layer, repeat the previous steps for mixing, pouring, and baking the PDMS.
Remove the PDMS sheet from the leveling box and cut individual membranes that match the dimensions of the well and microchannel layer. Use calipers to measure the membrane thickness at the center and discard any membranes that are outside of the desired thickness, then carefully clean the membranes with packaging tape and place them on a piece of paraffin film. To make the PDMS lid layer, repeat the previous steps for mixing, pouring, and baking the PDMS.
After cutting individual lids to size, punch access holes through the PDMS with a one millimeter biopsy punch and then surface clean them with packaging tape. Activate the surfaces of layers one and two with a plasma cleaner for 30 seconds on a medium radio frequency power setting, then align the access holes in layer one with the microchannels in layer two and firmly press the two layers together. For the deep-well design, repeat this process with layers two and three using double-sided tape to attach the paraffin film of layer three to a flat surface during plasma treatment.
Trim off excess material from the PDMS membrane and carefully peel away the sheet of paraffin film from the bottom of the chip. Bake the chip at 65 degrees Celsius for 10 minutes to increase bond strength between the PDMS layers, then insert angle dispensing tips into the access holes in the lid and secure them with a two-part epoxy using a micropipette tip to apply the epoxy around each tip. After the epoxy has fully cured, surface clean the chip with 70%ethanol and transfer it to a biosafety cabinet.
Connect a five milliliter syringe to the dispensing tips with sterile silicone tubing and fill the entire chip with 70%ethanol for at least 30 seconds. Remove the ethanol from the chip and sterilize it with UV light overnight. On the next day, wash the chip three times with distilled water, fill it with water, and remove the tubing from the dispensing tips.
Incubate the chip for at least 48 hours at 37 degrees Celsius. Place a compression spring around the shaft of the platen and insert it into the central hole on the bottom of the base, then attach the dial block to the bottom of the base with four self-tapping screws. Place a second compression spring around the central screw.
Insert the screw into the hexagonal-shaped hole in the bottom of the dial and screw the assembly into the threaded hole in the center of the dial block. Screw four male-female standoffs into the bottom of the base. Remove slack from the device by turning the dial counterclockwise until the top of the platen is below the top of the base, then slowly turn the dial clockwise until the top of the platen is level with the top of the base.
Use a five milliliter syringe to remove the water from the deep-well chip and coat the bottom of the well with 200 microliters of type I collagen in acetic acid for an hour. Rinse the chip three times using DPBS with calcium and magnesium and place it into the chip holder. Seed the chip with MLO-Y4 osteocytes in MEM alpha medium supplemented with calf serum, FBS, and penicillin/streptomycin.
Remove the tubing from the dispensing tips, place the chip into a deep-well culture dish, and incubate the cells at 37 degrees Celsius and five percent carbon dioxide for 72 hours. After the incubation, attach sterile tubing to dispensing tips and use a five milliliter syringe to remove the spent culture medium from the chip, then slowly refill the chip with fresh medium. Place the chip holder into the rectangular inset on the top of the loading device base, feed the tubing through the slots located on the lid, and secure the lid to the base.
Apply load to the cells by turning the dial clockwise until the desired platen displacement is reached, then place the loading device into an empty P1000 micropipette tip box and incubate the cells with the load for 15 minutes. After the incubation, remove the load from the cells by turning the dial counterclockwise to the original starting position, then remove the lid from the device, place the chip holder into the deep-well culture plate, and incubate the cells for a 90-minute post load recovery period. Use a five milliliter syringe to remove the conditioned medium from the chip and remove the chip from the chip holder, then use a tapered spatula to break the bond between the PDMS lid and well layer.
The cells are now ready to be analyzed. The shallow-well configuration can be used for analyzing functional activity of osteoblasts and osteoclasts. Bone formation from preosteoblasts was quantified using alizarin red and von Kossa stains.
Bone resorption from osteoclasts was quantified using toluidine blue staining and scanning electron microscopy was used to verify the presence of resorption pits. The deep-well chip configuration was used to induce osteocyte mechanotransduction by stretching the cells via a static out of plane distension. Images of osteocyte seeded in chips demonstrate that the 48-hour incubation of the chip with distilled water is critical for maintaining cell viability in typical morphology.
During bouts of loading, osteocytes were exposed to a strain gradient induced on the PDMS membrane. The relationship between average equivalent strain and platen displacement for values between one and two millimeters was determined and a representative heat map of the induced strain was generated. Following loading, cell viability was analyzed with lactate dehydrogenase staining and the annexin V and dead cell assay.
Lactate dehydrogenase stain of stretched osteocytes showed a lighter staining near the outer edge of the well, which corresponds to the location of higher strain values. Our platform provides a high degree of versatility and can be used to investigate a variety of factors that regulate bone remodeling such as load-induced mechanotransduction, inflammation, and drug effects. The techniques presented here provide a foundation for developing a true bone organ on a chip that could greatly enhance our understanding of the multicellular intricacies that regulate bone health.
