Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

The overall goal of this procedure is to generate genetically modified organotypic skin cultures using revitalized human dermis to determine the impact of altered gene expression on epidermal growth and differentiation. This method can help answer key questions in the dermatology field, such as determining which genes are responsible for epi growth, differentiation, and progression to disease. The main advantage of this technique is that genetically altered human skin is generated within a few days, which is allows the study of human samples that are potentially clinically relevant.

Demonstrating the procedure will be jingly from my laboratory. If receiving frozen human skin, place the tissue in the tissue culture hood for three to five minutes to thaw. Once thawed, transfer the tissue into a sterile 50 milliliter conical tube or 125 milliliter bottle, depending on size.

Next, add four x penicillin and streptomycin in one XPBS until the container is 75%full. Cap the container and shake vigorously for five minutes. Remove the antibiotic solution and repeat this.

Step three more times after the last wash, transfer the tissue to a new container and add fresh antibiotic solution. Incubate this mixture in a tissue culture incubator at 37 degrees Celsius for two weeks to allow the separation of the dermis from the epidermis. When two weeks have elapsed, use forceps to peel away the epidermis from the dermis.

Discard the epidermis according to the institution's protocol for dealing with human tissue. Place the dermis in a container of antibiotic solution and wash with vigorous shaking as before. After the last wash, transfer the dermis to a new container of antibiotic solution and store at four degrees Celsius until ready to use the day before transfection seed phoenix cells in complete media into a six well plate at a density of 800, 000 cells per well.

On the day of transfection, mix six microliters of transfection reagent with 100 microliters of dmm. For each well to be transfected and incubate at room temperature For five minutes, add the mixture to a tube containing three micrograms of retroviral vector for each well to be transfected and incubate at room temperature for 30 minutes, add this entire mixture dropwise to each well of the phoenix cells and incubate the cells overnight the day after transfection. Remove the media from the phoenix cells and add two milliliters of fresh complete media on the same day.

Resuspend primary human keratinocytes in keratinocyte serum free medium with antibiotics and seed into six wall plates at a density of 75, 000 cells per well. The following day, harvest the media which now contains viral particles from the transfected Phoenix cells. Pass the media through a 0.45 micron filter using a syringe to remove any contaminating cells.

Add five micrograms per milliliter of hexa dimethyl bromide to the filtered virus containing media to help mediate the infection process. Then pipette two milliliters of the virus onto the keratinocytes plated the previous day. Next, spin the six wall plates in a centrifuge at 200 Gs for one hour at room temperature.

After the spin, remove and discard the media containing virus. Wash the cells once with one XPBS and then add fresh K-C-S-F-M the following day. Infect the same batch of keratinocytes with virus using the procedure just shown.

To make organotypic cassettes. Use a cautery to remove a one centimeter by one 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 five minutes for the nail polish to dry.

Finally, flip the cassette over and place the cassette into a six centimeter dish with a cassette resting on the pegs. Retrieve the dermis from four 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 two 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 is set with the top of the dermis facing up.

Use forceps to flip the entire organotypic cassette containing the dermis. Add five drops of freshly ough 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 five minutes, use forceps to flip the cassette back over, and then add four milliliters of KGM to the six centimeter dish.

Next, after harvesting and counting the genetically modified keratinocytes Resus suspend 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 five. Tissue can be harvested up to 14 days after plating. This image shows hematin and eosin staining of organotypic.

Skin cultures regenerated human skin contains both the epidermis and dermis. The epidermis is fully stratified and differentiated with four distinct layers, including the undifferentiated basal layer and the three differentiated layers including the stratum, spinosum, ranum, and cornium. These immunofluorescence images demonstrate that overexpression of SNA i two as shown on the right inhibits epidermal differentiation compared to control tissue shown on the left staining for the differentiation.

Protein keratin one or K one is shown in green and nuclei is marked in blue. RTQ PCR R analysis to measure mRNA levels of SNAI two in control LAC Z and S NNA I two over expressing tissue demonstrates greater than 50 fold expression of SNAI two relative to control. Finally, this RTQ PCR R analysis demonstrates that overexpression of SNA I two results in loss of expression of differentiation induced structural genes such as TGM one and SPRR one A.Following this procedure.

Other methods like chipsy, immunofluorescence, RN, ae, or western blotting can be performed in order to determine the mechanisms of epidural growth and differentiation. This technique paved the way for researchers in the field of dermatology to explore how alter gene function impacts skin homeostasis. After watching this video, you should have a good understanding of how to generate genetically modified organic human skin cultures using keratinocytes and dev human dermis.