To begin, mix water, 10 times concentrated PBS, and one molar sodium hydroxide in a tube. Next, pipette 500 microliters of the appropriately dense dermal cell solutions to a two milliliter tube. Centrifuge the solution at 300g for three minutes at room temperature.
Once centrifugation is complete, pipette out the supernatant and gently resuspend the cells in a mixture of water, PBS, and sodium hydroxide. Now, add 200 microliters of the collagen solution to the mixture. Pipette the suspension to mix it well.
Next, pipette 500 microliters of the prepared mixture into an insert in a 24 well plate. For a model without the stratum corneum, pipette 500 microliters of the mixture to a well without an insert. After incubating the plate for 10 minutes at room temperature, transfer it to an incubator for 30 minutes.
Pipette 500 microliters of PBS buffered into each well to rinse the hydrogel surface. Next, mix 200 microliters of the keratinocyte suspension with an equal volume of melanocyte suspension in the supplemented DMEM medium. Gently pipette 500 microliters of the total cell suspension into each well.
Incubate the plates at 37 degrees Celsius for two to five days. Replace the medium every 48 hours and use an optical microscope to monitor the cell growth. An equivalent 3D model with distinguishable dermis and epidermis was created, which could be monitored in real time.
Keratinocytes in different stages of cell growth were observed, which are indicators of a living equivalent. Mast cells with recognizable granules were also seen.
