1. Preparation of acellular layer:

1. Preparation of acellular layer: Mix the following reagents in a 50 mL tube on ice: 0.59 mL 10X EMEM, 50 μl 200mM L-glutamine, 0.6 mL FBS, 120 μl 7.5% Sodium bicarbonate and 4.6 mL bovine collagen I. Add 1 mL of the mixture into each insert of tissue culture trays. Incubate for 30 min at room temperature and allow the gel to solidify. Color of the mixture should be from straw-yellow to pink.
2. Trypsinize human fibroblasts from culture flasks with 0.25% trypsin/EDTA, add DMEM containing 10% FBS to neutralize. Collect cells by centrifugation and resuspend 0.45 x 106 cells in 1.5 mL DMEM with 10% FBS. For dermal stem cells, collect 6600 dermal spheres (when dermal stem cells grow in stem cell medium, they form spheres in a similar manner to neurospheres) by tapping flask to detach the spheres,
centrifugation.
3. Preparation of cellular layer: Mix the following in a 50 mL tube on ice: 1.65 mL 10X EMEM, 150 μl 200 mM L-glutamine, 1.85 mL FBS, 350 μl 7.5% Sodium bicarbonate, 14 mL bovine collagen I and 1.5 mL fibroblasts suspension from step 2 (alternatively, use combination of 0.45 x 106 fibroblasts and 6600 dermal spheres in 1.5 mL of skin reconstruct medium I for dermal stem cell reconstructs), mix well. Add 3 mL of the mixture to each acellular layer-coated insert. Incubate for 45 min at 37 °C in a 5 % CO2 tissue culture incubator. Color of the mixture should be from straw yellow to pink. After the gel solidifies, add DMEM containing 10% FBS (2 mL inside and 10 mL outside of insert in each well of skin reconstruct trays). Incubate for 4 days and make sure that the gel contracts.
4. Aspirate medium from both inside and outside of each insert. Add washing medium (HBSS with 1% dialyzed FBS) 2 mL to the inside and 10 mL to the outside of insert in order to wash off regular serum. Incubate for 1 hour at 37 °C.
5. Preparation of skin reconstruct medium:
1. For normal melanocyte and dermal stem cell reconstructs: To make basic medium (500 mL), mix the following reagents: 490 mL keratinocyte serum-free medium, 1.8 mL bovine pituitary extract, 10 mL dialyzed fetal bovine serum, 500 μl of 10 μg/mL SCF, 562.5 μl of 4 μg/mL bFGF and 500 μl of 264 μg/mL ET-3. Medium I: Add 10 μl EGF of 100 μg/mL to 100 mL basic medium. Medium II: Add 2 μl EGF to 100 mL basic medium. Medium III: Add 720 μl CaCl2 of 1 M to 300 mL basic medium.
2. For melanoma skin reconstruct: To make Medium I (100mL), mix the following reagents: 72.5 mL of DMEM, 24 mL of F12 (HAM's), 2
mL L-glutamine of 200 mM, 200 μl hydrocortisone of 269 μg/mL, 200 μl ITES of 500X, 200 μl O-phosphorylethanolamine of 0.05 M, 200 μl adenine of 90 mM, 200 μl Progesterone of 2 nM, 240 μl CaCl2 of 1 M, 200 μl Triiodothyronine of 10 nM and 100 μl of chelexed


newborn calf serum (dissolve 3 g of Chelex 100 in 100 mL of serum, stir 1.5 hours at 4 °C, filter sterilize). To make Medium II (100mL), mix the following reagents: 72.5 mL of DMEM, 24 mL of F12 (HAM's), 2 mL L-glutamine of 200 mM, 200 μl hydrocortisone of 269 μg/ mL, 200 μl ITES of 500X, 200 μl O-phosphorylethanolamine of 0.05 M, 200 μl adenine of 90 mM, 200 μl Progesterone of 2 nM, 240
μl CaCl2 of 1 M, 200 μl Triiodothyronine of 10 nM and 100 μl newborn calf serum. To make Medium III (300mL), mix the following reagents: 142.5 mL of DMEM, 142.5 mL of F12 (HAM's), 6 mL of L-glutamine of 200 mM, 600 μl hydrocortisone of 269 μg/mL, 600 μl ITES of 500X, 600 μl O-phosphorylethanolamine of 0.05 M, 600 μl adenine of 90 mM, 600 μl Progesterone of 2 nM, 720 μl CaCl2 of 1 M, 600 μl Triiodothyronine of 10 nM and 6 mL newborn calf serum.

6. Trypsinize human keratinocytes from culture flasks with 0.05% trypsin/EDTA, neutralize trypsin with soy bean trypsin inhibitor (250 mg/L in 1x phosphate buffered saline), spin down, resuspend a cell pellet at 4.17 x106 cells/mL in skin reconstruct medium I.
7. Trypsinize human melanocytes or melanoma cells from culture flasks with 0.05% trypsin/EDTA, neutralize trypsin with soy bean trypsin inhibitor, spin down, resuspend a cell pellet at 0.83 x106 cells/mL in skin reconstruct medium I.
8. Remove washing medium from both inside and outside of each insert.
9. Add skin reconstruct medium I (1.5 mL to the inside and 10 mL to the outside of each insert).
10. Take 600 μl cell suspension of keratinocytes and 600 μl cell suspension of melanocytes or melanoma cells, mix well. Dispense 200 μl mixed cell suspension drop by drop to the inside of each insert. For dermal stem cell reconstruct, use 100 μl of keratinocyte suspension only. Incubate for 2 days at 37 °C.
11. Aspirate skin reconstruct medium I from both the inside and the outside of each insert. Add skin reconstruct medium II (2 mL to the inside and 10 mL to the outside). Incubate for another 2 days at 37 °C.
12. Aspirate skin reconstruct medium II from the inside and the outside of each insert, add 7.5 mL skin reconstruct medium III to only the outside. From this point, the surface of reconstructs starts being exposed to