Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

Our challenge is to test ingredients, cytotoxicity prior to cosmetic or medical use. The commercial 3D skin models limited to around free cell types don't reflect real skin complexity, and skin has diverse cell types with essential functions, posing a challenge in accurately assessing ingredient impact. Our findings indicate that the limiting cell adhesion method produce more well-shaped spheres compared to the commonly used methods.

Additionally, it demands less manipulation, ensuring greater sphere stability. In our skin equivalent, we use up to four types of skin cells, unlike the commercial models that typically incorporate a maximum of three cell types. Common 2D cell models lack accuracy in representing real interaction, while commercial 3D skin equivalence offer better tissue representation, they cost limits widespread use.

Consequently, our self-established models allow for initial cost-effective experiments on a more realistic platform. Our protocols outline affordable, straightforward ways to create customized 3D skin models, encompassing spheres and equivalence. In contrast to commercial options, our models offer adjustable appearance and composition tailored to the studies requirements, The methods accessible, not demanding specialized skills or equipment, making them replicable even by novice researchers.

Our aim is to highlight the simplicity of creating various 3D cellular models. While emphasizing the value of certified commercial models in research, we stress the significance of using realistic models such as spheres and equivalence, showcasing how the self-made models can aid in pre-selecting the testing conditions before employing certified models. To begin, remove the respective medium from the culture flasks containing the keratinocytes, fibroblasts and melanocytes.

Gently wash the cells with five to 10 milliliters of PBS. Now add 0.5 to two milliliters of Trypsin-EDTA solution to the flask, depending on its size. Incubate the flask at 37 degrees Celsius.

Use a microscope to control the detachment of cells from the surface. Suspend the detached cells in double the volume of trypsin neutralizer to deactivate trypsin, then transfer the suspension into a 15-milliliter tube. Now pipette about 20 microliters of the skin cell suspension into a 1.5 milliliter tube.

With the help of a hemocytometer, count the cells. Next, centrifuge the tube at 300 G for five minutes at room temperature. Then pipette out most of the supernatant and resuspend the cell palate in the small amount of remaining liquid.

Add an equal volume of fresh medium to the resuspended cells. In a separate 15-milliliter tube, prepare cell suspension. The primary cells of keratinocytes, melanocytes, fibroblasts, and mast cells presented their typical morphology when grown in their respective media.

To begin the hanging drop method, pipette 20 microliters of a pre-prepared Skin Cell Suspension onto the lid of a Petri dish. Now cover the lid with the bottom half of the Petri dish and gently flip it to create the hanging drops. Add sterile water to the Petri dish to prevent evaporation of the full grown medium from drop droplets.

Incubate the droplets for 48 to 72 hours at 37 degrees Celsius. Next, fill the wells of a new plate with the full growth medium. Using sterile cut pipette tips, transfer the cell spheres to the wells of the multi-well plate.

Incubate the transferred spheres for one day in the multi-well plate at 37 degrees Celsius before experimentation. For the limiting cell adhesion method, first, add 100 microliters of 1%surfactant solution in PBS into each well of a U-Bottom plate. Next, incubate the plate with the solution at 37 degrees Celsius for 24 hours.

Now prepare the cell suspension and the desired Cell Density. Pipette out the surfactant into solution from the wells, then seed 50 microliters of the cell suspension into each well. Finally, incubate the plate at 37 degrees Celsius for 24 hours to reach cell aggregates.

Spheres consisting of 10, 000 cells were easier to manipulate as they were visible in the wells. The bigger spheres showed decreased stability. Different cell types formed spheres of different colors.

The perfectly rounded spheres were prone to losing their shape during the transfer in the hanging drop method. Accurate handling was required to reduce the deformation resulting in intact cell aggregates. Inaccuracy and inexperience resulted in sphere damage.

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 300 G for three minutes at room temperature.

Once centrifugation is complete, pipette out the supranatant 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 buffer 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 the 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.