Organotypic 3D Skin Culture: A Cassette-based In Vitro Culture Technique to Regenerate Human Epidermis on Acellular Dermis

- Organotypic skin cultures help generate reconstructed skin tissue with typical epidermal differentiation and stratification characteristics. They mimic the functional and physiological in vivo tissue architecture.

To generate an organotypic skin, first, assemble a pre-cut, dead, de-epidermized dermis onto a customized cassette with its top layer facing upwards. This dermis forms the substrate to aid skin regeneration.

Invert the cassette assembly to expose the bottom layer of the dermis. Pipet an artificial extracellular matrix over the bottom layer. Allow the matrix to solidify and provide structural support to the dermis.

Invert the assembly back to its initial position. Add the desired volume of keratinocyte growth media to immerse just the bottom layer of the dermis. Add keratinocyte suspension onto the upper layer of the dermis to mimic the typical skin tissue architecture and incubate.

During culture, the growth media diffusing through the dermis supplements keratinocyte proliferation. Additionally, the disposition of these proliferating keratinocytes at the air-liquid interface promotes their differentiation. The process regenerates a fully stratified epidermis over the devitalized dermal layer, producing an organotypic skin reconstruct. In the following protocol, we show the generation of human organotypic skin using genetically modified primary human keratinocytes and devitalized human dermis.

- To make organotypic cassettes, use a cautery to remove a 1 centimeter by 1 centimeter square from the center of the lid of a 3.5 centimeter tissue culture dish. Attach square pegs to the bottom of the cassette using clear nail polish and allow 5 minutes for the nail polish to dry.

Finally, flip the cassette over and place the cassette into a 6-centimeter dish with a cassette resting on the pegs. Retrieve the dermis from 4 degrees Celsius storage and transfer it to a container of keratinocyte growth medium to wash.

After repeating the wash, place the dermis in fresh KGM and incubate at 37 degrees Celsius for 2 days. Following the incubation, use a scalpel to cut the dermis into 1.5 centimeter by 1.5 centimeter-sized pieces. Then, place onto the square hole of the organotypic cassette with the top of the dermis facing up.

Use forceps to flip the entire organotypic cassette containing the dermis. Add five drops of freshly thawed extracellular matrix to the bottom of the dermis and shake slightly to ensure even distribution across the dermis. After allowing the extracellular matrix to solidify for 5 minutes, use forceps to flip the cassette back over and then add 4 milliliters of KGM to the 6-centimeter dish.

Next, after harvesting and counting the genetically modified keratinocytes, resuspend 0.5 to 1 million cells in 90 microliters of KGM and dispense the cells onto the dermis. Incubate the co-culture at 37 degrees Celsius.

Change the media on the cultures every other day. Full stratification and differentiation of the epidermis usually occurs after day 5. Tissue can be harvested up to 14 days after plating.