The overall goal of this 3D organotypic melanoma spheroid skin model is to recapitulate both the architecture and multicellular complexity of an organ or tumor in vivo while accommodating systematic experimental intervention. This method can help to answer key questions in the field of melanoma research such as tumor-host interaction and therapeutic intervention. The main advantage of this technique is that we can mimic the 3D architecture of non vascularized metastases in vivo.

The implications of this technique extend to what therapy of melanoma because it reflects the tumor heterogeneity. In general, individuals new to this method may struggle because the quality of the primary cells is decisive and no antibiotics are used during the cell process. The implications of this technique extend to what therapy of melanoma because it reflects tumor heterogeneity.

Visual demonstration of this method is critical because proper handling of the 3D melanoma skin model is very important. To begin, culture melanoma cells 451LU using RPMI medium containing 10%FCS, according to general cell culture protocols. To generate melanoma spheroids, wash the cultured melanoma cells in PBS.

Then add five milliliters of one times trypsin EDTA in PBS. Incubate for three to five minutes at room temperature. Add five milliliters of RPMI containing 10%FCS to neutralize the trypsin.

Then transfer the cell suspension to a centrifuge tube. Centrifuge at 200 times g for five minutes to harvest the cells. Re suspend the cell pellet in RPMI containing 10%FCS.

Next, count the cells, and dilute the cell suspension to a final concentration of 10, 000 cells per milliliter. Use an electronic multi pipette to make 40 25 microliter spots of cell suspension on the inner surface of the lid of a sterile non-adhesive 10 centimeter cell culture dish. Next, turn the lid over with a fast, smooth movement and place it on the cell culture dish containing five milliliters of PBS.

Culture the hanging drop dishes at 37 degrees Celsius in 5%carbon dioxide for 10 to 14 days. Five days after the initial drop spotting, use an electronic dispenser to add 10 microliters of RPMI containing 10%FCS to each drop. After 10 to 15 days, harvest the spheroids by gently rinsing them off the lid with PBS.

Collect the spheroids in a fresh non-adhesive cell culture dish. To generate the dermal counterpart of the skin reconstructs, place eight micrometer pore size membrane inserts in a 24 well cell culture plate. For each insert, resuspend 100, 000 fibroblasts in gel neutralization solution.

Next, quickly mix the fibroblast suspension with collagen type one at a ratio of one to three in a final volume of 500 microliters without forming bubbles. Then use a pipette to add the suspension to each insert. Inside a sterile hood, place the inserts at room temperature without medium for 30 minutes to allow the dermal gels to settle.

Next, cover the gels with the DMEM and incubate overnight at 37 degrees Celsius. After the overnight incubation, remove the DMEM from the dermal gels and dequilibrate them with EGM for two hours at 37 degrees Celsius to generate the epidermal counterpart of the skin reconstructs. Next use a pipette to remove the EGM from the gels.

Then carefully seed 100, 000 keratinocytes, resuspended in 100 microliters of EGM on top of each gel. Incubate the reconstructs for one and a half hours at 37 degrees Celsius to allow the keratinocytes to adhere to the dermal gel. Using a pipette tip, carefully remove the residual gel from the insert wall.

Next, cover each reconstruct with 800 microliters of EGM and culture for seven days at 37 degrees Celsius in 5%carbon dioxide. Carefully remove residual gel from the inside wall with a small, wide pipette tip. On day eight, place the inserts into separate wells of a six well plate.

Next add 1.2 to 1.4 of metastatic melanoma medium to each well to cover only the base of the skin reconstruct. Then, incubate for 10 to 17 days at 37 degrees Celsius in 5%carbon dioxide. Add only 1.2 to 1.4 mL of metastatic melanoma medium to each well so that the skin reconstruct is supplied with medium from the bottom of the well but it's not covered with medium.

To integrate melanoma spheroids into the dermal counterpart of the full skin reconstructs, use PBS to carefully rinse the spheroids off the lid of a hanging drop cell culture dish. For each full skin reconstruct, collect 10 to 20 spheroids in a sterile, nonadhesive cell culture dish. Use a Pasteur pipette to carefully remove excess PBS.

Next, aspirate the spheroids in a minimal volume of EGM. Then transfer them to the required volume of gel neutralization solution, containing 100, 000 fibroblasts. For each insert, mix the spheroid suspension with collagen type one at a ratio of one to three in a final volume of 500 microliters.

Finally, generate organotypic full skin reconstructs as outlined in the previously described protocol. Normal human skin is compared to the skin reconstruct to validate the 3D organotypic skin reconstruct. Hematoxylin eosin staining of the paraffin sections shows that the dermis and epidermis of the skin reconstruct are comparable to those in normal skin.

Normal human skin and skin reconstruct are immunohistochemically analyzed to compare the epidermal differentiation. Immunostaining against epidermal stratification markers, namely keratin 14, keratin 10 and involucrine and filagrin indicates similar levels of keratinaside differentiation between the skin reconstruct and normal skin. Immunostaining against laminin five indicates that the basal lamina is formed to physiologically connect the epidermal and dermal counterparts of the skin reconstructs, similar to normal skin.

Hematoxylin eosin staining of the paraffin section of melanoma spheroid skin reconstruct indicates that the cellular distribution of melanoma spheroids is similar to that of the non vascularized human melanoma skin metastases in vivo. Histological examination reveals the presence of a peripheral proliferating subpopulation and central necrotic subpopulation of the melanoma cells. Once mastered, this technique could be done in about 40 days, if it is performed properly.

While attempting this procedure, it is important to use primary cells of best quality. After it's development this technique paved the way for melanoma researchers to explore the responsiveness in vitro in an in vivo mimicking environment. After watching this video, you should have a good understanding how to generate a human 3D organotypic melanoma skin model.