The overall goal of this procedure is to generate a 3D human skin reconstruct for studies of melanocyte, melanoma and skin stem cell biology. This is accomplished by first coating an insert of tissue culture trays with neutralized bovine collagen. The second step is to make the cellular dermal compartment from fibroblasts and collagen.
Next, make the epidermal compartment from keratinocytes mixed with melanocytes or melanoma cells. The final step of the procedure is to harvest a skin reconstruct at day 18 and to graft a skin reconstruct onto a mouse. Ultimately, immunohistochemical and immunofluorescent analyses of the graft show the architecture of normal skin containing melanocytes, the migration and the differentiation of normal stem cells and stage specific melanoma phenotypes.
We developed the skin reconstruction model 20 years ago because we early on in our studies found that normal melanocytes or melanoma cells that are cultured on a dish behave very different from cells that are in a tissue context. So the skin reconstruction model is ideal for us to see how cells move from one compartment in the skin to any other Place. The previously prepared acellular layer collagen mixture on ice, and then add one milliliter of the straw yellow solution into each insert of a six.
Well tissue culture tray incubate the tray for 30 minutes at room temperature to allow the gel to solidify. During this time, the solution should change in color from yellow to pink while the gel is solidifying trypsin eyes, human fiberblast from their culture flask with 0.25%tripsin EDTA after five minutes. Add DMEM containing 10%FBS to neutralize the ization process.
Collect the detached cells by centrifugation for five minutes at 200 G at room temperature, and then resuspend the cells at 4.5 times 10 to the fifth cells per 1.5 milliliters. DMEM supplemented with 10%FBS. Next, fill a 50 milliliter tube with the previously prepared cellular layer collagen mixture and place the tube on ice.
Add 1.5 milliliters of the fibroblast suspension to the tube and mix.Well. Add three milliliters of the straw yellow cellular layer solution to each acellular layer coated insert. Then incubate the tissue culture tray for 45 minutes at 37 degrees Celsius in a 5%carbon dioxide tissue culture incubator during the incubation period.
The color of the cellular layer solution should also change from yellow to pink. After the top layer gel solidifies, add two milliliters of DMEM containing 10%FBS to the inside of the insert in the tissue culture tray, and 10 milliliters to the outside of the insert. Incubate the tissue culture trays for four days.
Checking on the fourth day that the gel contracts on the fourth day of incubation aspirate the medium from both the inside and outside of each insert. Add two milliliters of wash medium to the inside and 10 milliliters to the outside of the inserts to wash off the regular serum. Then incubate the trays for one hour at 37 degrees Celsius for generation of the epidermis.
First trypsin is human keratinocytes. Then after five minutes, use soybean trypsin inhibitor to neutralize the trypsin after spinning down the detached cells at 200 G for five minutes. Reus suspend the cell pellet at 4.17 times 10 to the six cells per milliliter in skin.
Reconstruct medium one after the keratinocytes have been harvested. Trypsin ice human melanocytes for one to two minutes. Again, neutralize the trypsin with soybean trypsin inhibitor.
Then after spinning down the cells under the same conditions as before, resuspend the melanocyte pellet at 8.3 times 10 to the fifth cells per milliliter in skin. Reconstruct medium one as well. Next, remove the washing medium from both the inside and the outside of each insert of a dermal layer prepared tissue culture tray.
Replace the washing medium by adding 1.5 milliliters of skin, reconstruct medium, one to the inside and 10 milliliters to the outside of each insert. Then mix 600 microliters of the keratinocyte cell suspension with 600 microliters of the melanocyte suspension. Dispense 200 microliters of the mixed cell suspension.
Drop by drop to the inside of each insert. Incubate the tissue culture tray for two days at 37 degrees Celsius. After incubating the cells, aspirate the skin, reconstruct medium one from both the inside and the outside of each insert, and then add two milliliters of skin, reconstruct medium, two to the inside and 10 milliliters to the outside of the insert.
Then incubate the reconstructs for another two days at 37 degrees celsius. After the second two day incubation aspirate the skin, reconstruct medium two from the inside and the outside of each insert. Now add 7.5 milliliters of the skin, reconstruct medium three to only the outside of the inserts.
Place the reconstructs back in the incubator and change the skin. Reconstruct medium three every other day until day 18. On day 18 of the incubation aspirate the media from the inside and the outside of the inserts of a skin reconstruct loaded tissue culture tray.
Remove the inserts from the tray with forceps. Cut out the construct including the polycarbonate filter by tracing a circle close to the edge with a scalpel blade. Then place the reconstruct in the filter on a hard surface and cut them in half with the blade.
Place one half of the reconstruct on a black TBS biopsy paper, and then place the paper into a histology cassette. Soak the whole cassette in 10%formalin for more than four hours. Then place the cassette in 70%ethanol and store it at four degrees Celsius until you are ready to process the reconstruct for paraffin embedding.
Place the other half of the reconstruct in 50%sucrose at four degrees Celsius for one to two hours. Then change the sucrose to two molar and then store it at four degrees Celsius for another one to two hours. Fill a base mold halfway with optimal cutting temperature, freezing media or OCT.
Avoid any bubbles. Grip the edge of the reconstruct with forceps and remove the reconstruct from the sucrose. Place it on Kim wipes until the sucrose is absorbed.
Using forceps and a spatula, transfer the reconstruct into the boat on top of the OCT. Cover the top of the reconstruct with more OCT until the boat is completely full. Again, avoiding bubbles.
Place the boat evenly on crushed dry ice to allow the OCT to freeze completely. Then wrap the base mold and foil and store it at minus 70 degrees Celsius until you are ready to cut it with a cryostat. To prepare for grafting a skin reconstruct on a mouse, fill a container with liquid nitrogen and add five milliliters of DMEM to a 50 milliliter tube.
Fill a beaker with 600 milliliters of tap water and place the beaker on top of a hot plate to warm. Then select a female skid hairless outbred mouse around six weeks in age. After anesthetizing the mouse with isof fluorine.
Use a nose cone to maintain the anesthesia throughout the procedure. Apply sterile ophthalmic lubricant and provide heat support to prevent hypothermia. Next, clean the mouse skin with compound benzoin tincture and alcohol prep swabs.
Mark a line on top of the mouse back to keep the same position for later suturing. Cut a round wound bed on the mouse back using iris scissors and forceps, and then cover the wound bed with sterile gauze sponges. Put the round mouse skin in the 50 milliliter tube of DMEM.
Immerse the tube in a liquid nitrogen container until all the media and tissue are totally frozen. Next, defrost the tube in a beaker of hot water until totally thawed. Repeat this, freeze and thaw procedure three times.
Use a surgical blade to detach a skin reconstruct from an insert. Remove the membrane very carefully and then transfer the skin reconstruct to the top of the mouse wound bed. Now, place the mouse skin on top of the skin reconstruct.
Make sure that the first suture is on top of the previously labeled marker and that the second is on the bottom. Then use two more sutures on the sides of the skin reconstruct to fix the mouse skin. Clean the mouse skin after grafting, and then put the mouse into a new cage.
At day 18. The epidermis of skin reconstructs is composed of stratified keratinocyte layers, the undifferentiated basal layer and sequentially differentiated layers are vertically oriented. Staining the section of reconstructs with the melanocytic marker.
S 100 as indicated by the black arrows, shows that melanocytes are aligned in the basal layer of the epidermis and communicate with multiple keratinocytes through dendrite extensions. The dermal compartment contains fibroblasts embedded in a collagen type one matrix deposited collagen four indicates the basement membrane, which separates the epidermis from the dermis. The following figures illustrate how multiple layers of keratinocytes in the epidermis are developed.
When dermal stem cells labeled with a GFP lentiviral vector are embedded with fibroblasts. In a collagen type one matrix, they migrate to the epidermis and differentiate into melanocytes at day five. After seeding keratinocytes, single cells start migrating out from spheres.
Note that the epidermis is still composed of a single layer. At day eight. A few cells reach the epidermis dermis interface.
By day 10 GFP positive cells are tightly aligned at the basement membrane position as can be seen here. Migrated GFP positive cells in the epidermis express the melanocytic marker HMB 45. When different stages of melanoma cell lines are incorporated in skin reconstructs, the behavior of the cells reflects their in vivo characteristics.
Here, normal melanocytes and melanoma cells grown in 2D cultures can be seen in this figure. The location and the growth rate of normal melanocytes are tightly controlled in skin reconstructs as seen here, radial growth phase, primary melanomas proliferate predominantly in the epidermis, whereas vertical growth phase melanomas grow invasively into the dermis as seen in this figure. Finally, the way that metastatic melanomas aggressively invade deep into the dermis can be seen here.
Our studies in normal skin provide the foundation for a better knowledge of melanoma development and progression.
