preparation of acellular layer : mix the following reagents in a 50 ml tube on ice : 0.59 ml 10x emem , 50 l 200 mm l - glutamine , 0.6 ml fbs , 120 l 7.5% sodium bicarbonate and 4.6 ml bovine collagen i. add 1 ml of the mixture into each insert of tissue culture trays . 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 10 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 . preparation of cellular layer : mix the following in a 50 ml tube on ice : 1.65 ml 10x emem , 150 l 200 mm l - glutamine , 1.85 ml fbs , 350 l 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 10 fibroblasts and 6600 dermal spheres in 1.5 ml of skin reconstruct medium i for dermal stem cell reconstructs ) , mix well . incubate for 45 min at 37 c in a 5 % co2 tissue culture incubator . 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 ) . 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 . preparation of skin reconstruct medium : 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 l of 10 g / ml scf , 562.5 l of 4 g / ml bfgf and 500 l of 264 g / ml et-3 . medium i : add 10 l egf of 100 g / ml to 100 ml basic medium . medium iii : add 720 l cacl2 of 1 m to 300 ml basic medium.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 l hydrocortisone of 269 g / ml , 200 l ites of 500x , 200 l o - phosphorylethanolamine of 0.05 m , 200 l adenine of 90 mm , 200 l progesterone of 2 nm , 240 l cacl2 of 1 m , 200 l triiodothyronine of 10 nm and 100 l 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 l hydrocortisone of 269 g / ml , 200 l ites of 500x , 200 l o - phosphorylethanolamine of 0.05 m , 200 l adenine of 90 mm , 200 l progesterone of 2 nm , 240 l cacl2 of 1 m , 200 l triiodothyronine of 10 nm and 100 l 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 l hydrocortisone of 269 g / ml , 600 l ites of 500x , 600 l o - phosphorylethanolamine of 0.05 m , 600 l adenine of 90 mm , 600 l progesterone of 2 nm , 720 l cacl2 of 1 m , 600 l triiodothyronine of 10 nm and 6 ml newborn calf serum . 
  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 l of 10 g / ml scf , 562.5 l of 4 g / ml bfgf and 500 l of 264 g / ml et-3 . medium i : add 10 l egf of 100 g / ml to 100 ml basic medium . medium iii : add 720 l cacl2 of 1 m to 300 ml basic medium . 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 l hydrocortisone of 269 g / ml , 200 l ites of 500x , 200 l o - phosphorylethanolamine of 0.05 m , 200 l adenine of 90 mm , 200 l progesterone of 2 nm , 240 l cacl2 of 1 m , 200 l triiodothyronine of 10 nm and 100 l 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 l hydrocortisone of 269 g / ml , 200 l ites of 500x , 200 l o - phosphorylethanolamine of 0.05 m , 200 l adenine of 90 mm , 200 l progesterone of 2 nm , 240 l cacl2 of 1 m , 200 l triiodothyronine of 10 nm and 100 l 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 l hydrocortisone of 269 g / ml , 600 l ites of 500x , 600 l o - phosphorylethanolamine of 0.05 m , 600 l adenine of 90 mm , 600 l progesterone of 2 nm , 720 l cacl2 of 1 m , 600 l triiodothyronine of 10 nm and 6 ml newborn calf serum . 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 x10 cells / ml in skin reconstruct medium i. 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 x10 cells / ml in skin reconstruct medium i. remove washing medium from both inside and outside of each insert . add skin reconstruct medium i ( 1.5 ml to the inside and 10 ml to the outside of each insert ) . take 600 l cell suspension of keratinocytes and 600 l cell suspension of melanocytes or melanoma cells , mix well . dispense 200 l mixed cell suspension drop by drop to the inside of each insert . for dermal stem cell reconstruct , use 100 l of keratinocyte suspension only . 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 ) . 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 . harvest skin reconstruct at day 18 : aspirate media from the inside and the outside of inserts.remove inserts from tray with forceps.cut out the reconstruct ( including the polycarbonate filter ) by tracing a circle close to the edge with a scalpel blade.cut the reconstruct and the filter in half on a hard surface . cut out the reconstruct ( including the polycarbonate filter ) by tracing a circle close to the edge with a scalpel blade . for paraffin sections : place a half of the reconstruct in a histology cassette between 2 black tbs biopsy papers and soak the whole cassette in 10% formalin for more than 4 hours . then place the cassette in 70% ethanol and store it at 4 c until you are ready to process the reconstruct for paraffin embedding . for frozen sections : place the other half of reconstruct in 50% sucrose at 4 c for 1 - 2 hours , then change the sucrose to 2 m and store it at 4 c for another 1 - 2 hours.dispense optimal cutting temperature freezing media ( oct ) into a disposable base mold so that it fills about 50% of the boat . avoid any bubbles in the oct.grab the edge of the reconstruct with forceps and remove the reconstruct from the sucrose . place it on kimwipes until the kimwipes absorb the sucrose.transfer the reconstruct into the base mold on top of the oct , using forceps and a spatula . cover the top of the reconstruct with more oct until the base mold is completely full . make sure you do not have any bubbles in the oct which will make cutting difficult.place the base mold evenly on crushed dry ice , and allow the oct to freeze completely.wrap the base mold in tin foil and store it at -70 c until you are ready to cut it with a cryostat . place the other half of reconstruct in 50% sucrose at 4 c for 1 - 2 hours , then change the sucrose to 2 m and store it at 4 c for another 1 - 2 hours . dispense optimal cutting temperature freezing media ( oct ) into a disposable base mold so that it fills about 50% of the boat . grab the edge of the reconstruct with forceps and remove the reconstruct from the sucrose . transfer the reconstruct into the base mold on top of the oct , using forceps and a spatula . cover the top of the reconstruct with more oct until the base mold is completely full . make sure you do not have any bubbles in the oct which will make cutting difficult . place the base mold evenly on crushed dry ice , and allow the oct to freeze completely . wrap the base mold in tin foil and store it at -70 c until you are ready to cut it with a cryostat . graft a skin reconstruct on a mouse : prepare a 50 ml falcon tube with 5 ml of dmem ( for freezing and defrosting mouse skin).use a female scid hairless outbred mouse around 6 weeks.the mouse is anesthetized with isoflurane ( ez anesthesia system ) : initial anesthesia is performed in the induction chamber with 4% isoflurane ( before mouse is moved to the surgical bed ) and maintain anesthesia through a nosecone breather during procedure ( isoflurane : 1.5 - 2% ) . heat support by a heated pad to prevent hypothermia and sterile ophthalmic lubricant applied to prevent ocular injury during anesthesia.put 600 ml water in a beaker and leave on top of a hot plate to keep water temperature between 40 - 60 c.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 suturing later.cut a round wound bed about 1.2 cm in diameter on the mouse upper back using iris scissors and forceps . cover the wound bed with sterile gauze sponges . put the round mouse skin in 5 ml of dmem , then leave in liquid nitrogen container until it totally frozen . defrost the tube in hot water on top of a hot plate until totally thawed . repeat 3 times.detach the skin reconstruct from the transwell using surgical blade and remove the membrane very carefully , transfer the skin reconstruct ( epidermis side up ) on top of the wound bed.place the mouse skin ( epidermis side up ) on top of the skin reconstruct.make the first suture on the marker previously labeled , the second on the bottom , then two more sutures on sides to fix the mouse skin.clean mouse skin after grafting . remove nosecone and keep mouse on the heated pad until awake and ambulatory.put the mouse into a new cage.post operative analgesia : meloxicam ( 1 mg / kg ) by subcutaneous injection immediately after surgery , 24 hours and 48 hours after surgery . prepare a 50 ml falcon tube with 5 ml of dmem ( for freezing and defrosting mouse skin ) . use a female scid hairless outbred mouse around 6 weeks . the mouse is anesthetized with isoflurane ( ez anesthesia system ) : initial anesthesia is performed in the induction chamber with 4% isoflurane ( before mouse is moved to the surgical bed ) and maintain anesthesia through a nosecone breather during procedure ( isoflurane : 1.5 - 2% ) . heat support by a heated pad to prevent hypothermia and sterile ophthalmic lubricant applied to prevent ocular injury during anesthesia . put 600 ml water in a beaker and leave on top of a hot plate to keep water temperature between 40 - 60 c . 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 suturing later . cut a round wound bed about 1.2 cm in diameter on the mouse upper back using iris scissors and forceps . put the round mouse skin in 5 ml of dmem , then leave in liquid nitrogen container until it totally frozen . defrost the tube in hot water on top of a hot plate until totally thawed . detach the skin reconstruct from the transwell using surgical blade and remove the membrane very carefully , transfer the skin reconstruct ( epidermis side up ) on top of the wound bed . place the mouse skin ( epidermis side up ) on top of the skin reconstruct . make the first suture on the marker previously labeled , the second on the bottom , then two more sutures on sides to fix the mouse skin post operative analgesia : meloxicam ( 1 mg / kg ) by subcutaneous injection immediately after surgery , 24 hours and 48 hours after surgery . skin reconstructs with normal melanocytes and dermal stem cells the skin reconstructs are cultured in a special 6-well tray with inserts . the dermal compartment is cultured in dmem with 10% fbs for the first four days ( fig . the epidermis is exposed to the air at day 9 and this allows keratinocytes to differentiate ( fig . 1c ) . at day 18 , the epidermis of skin reconstructs is composed of stratified keratinocyte layers . staining the section of reconstructs with the melanocytic marker s100 shows that melanocytes are aligned in the basal layer of the epidermis and communicate with multiple keratinocytes through dendrite extensions ( fig . deposited collagen iv indicates the basement membrane , which separates the epidermis from the dermis ( fig . when dermal stem cells ( labeled with gfp lentiviral vector ) are embedded with fibroblasts in a collagen i matrix , they migrate to the epidermis and differentiate into melanocytes ( 1 ) , ( fig . skin reconstructs of melanoma tumors clinicopathological studies indicate that melanomas progress in a stepwise manner : common acquired nevi , dysplastic nevi , rgp ( radial growth phase ) melanomas , vgp ( vertical growth phase ) melanomas and metastatic melanomas ( 7 ) . different stages of melanoma cell lines are morphologically similar to each other in 2-d culture ( fig . 3a - d ) but when they are incorporated in skin reconstructs , the behavior of the cells reflects their in vivo characteristics . the location and the growth rate of normal melanocytes are tightly controlled in skin reconstructs ( fig . a. fibroblasts mixed with collagen are grown in dmem containing 10% fbs and form dermal compartment . b. keratinocytes and melanocytes are seeded on top of the dermis and grow in skin reconstruct medium . c. the epidermis is exposed to air at day 9 . d. h&e - stained skin reconstruct presents the epidermis comprised vertically , oriented basal layer , and sequentially differentiated stratified cell layers . e. s100-positive melanocytes ( black arrows ) are aligned at the basement membrane and communicate with multiple keratinocytes . f. collagen iv - staining indicates the basement membrane , which separates the epidermis from the dermis . all the stainings in d - f were performed on formalin - fixed , paraffin - embedded sections . dermal stem cells in skin reconstructs migrate to the epidermis and differentiate into melanocytes . at day 5 after seeding keratinocytes , single gfp - positive cells ( green ) the epidermis is still composed of a single layer . at day 8 , a few cells reach the epidermis - dermis interface . by day 10 the migrated gfp - positive cells in the epidermis express melanocytic marker hmb45 ( red , as indicated by white arrows ) . g. vgp melanoma wm793 cells invade into the dermis through basement membrane . h. metastatic melanoma 1205lu cells aggressively invade deep into dermis . preparation of acellular layer : mix the following reagents in a 50 ml tube on ice : 0.59 ml 10x emem , 50 l 200 mm l - glutamine , 0.6 ml fbs , 120 l 7.5% sodium bicarbonate and 4.6 ml bovine collagen i. add 1 ml of the mixture into each insert of tissue culture trays . 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 10 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 . preparation of cellular layer : mix the following in a 50 ml tube on ice : 1.65 ml 10x emem , 150 l 200 mm l - glutamine , 1.85 ml fbs , 350 l 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 10 fibroblasts and 6600 dermal spheres in 1.5 ml of skin reconstruct medium i for dermal stem cell reconstructs ) , mix well . incubate for 45 min at 37 c in a 5 % co2 tissue culture incubator . 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 ) . 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 . preparation of skin reconstruct medium : 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 l of 10 g / ml scf , 562.5 l of 4 g / ml bfgf and 500 l of 264 g / ml et-3 . medium i : add 10 l egf of 100 g / ml to 100 ml basic medium . medium iii : add 720 l cacl2 of 1 m to 300 ml basic medium.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 l hydrocortisone of 269 g / ml , 200 l ites of 500x , 200 l o - phosphorylethanolamine of 0.05 m , 200 l adenine of 90 mm , 200 l progesterone of 2 nm , 240 l cacl2 of 1 m , 200 l triiodothyronine of 10 nm and 100 l 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 l hydrocortisone of 269 g / ml , 200 l ites of 500x , 200 l o - phosphorylethanolamine of 0.05 m , 200 l adenine of 90 mm , 200 l progesterone of 2 nm , 240 l cacl2 of 1 m , 200 l triiodothyronine of 10 nm and 100 l 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 l hydrocortisone of 269 g / ml , 600 l ites of 500x , 600 l o - phosphorylethanolamine of 0.05 m , 600 l adenine of 90 mm , 600 l progesterone of 2 nm , 720 l cacl2 of 1 m , 600 l triiodothyronine of 10 nm and 6 ml newborn calf serum . 
  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 l of 10 g / ml scf , 562.5 l of 4 g / ml bfgf and 500 l of 264 g / ml et-3 . medium i : add 10 l egf of 100 g / ml to 100 ml basic medium . medium iii : add 720 l cacl2 of 1 m to 300 ml basic medium . 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 l hydrocortisone of 269 g / ml , 200 l ites of 500x , 200 l o - phosphorylethanolamine of 0.05 m , 200 l adenine of 90 mm , 200 l progesterone of 2 nm , 240 l cacl2 of 1 m , 200 l triiodothyronine of 10 nm and 100 l 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 l hydrocortisone of 269 g / ml , 200 l ites of 500x , 200 l o - phosphorylethanolamine of 0.05 m , 200 l adenine of 90 mm , 200 l progesterone of 2 nm , 240 l cacl2 of 1 m , 200 l triiodothyronine of 10 nm and 100 l 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 l hydrocortisone of 269 g / ml , 600 l ites of 500x , 600 l o - phosphorylethanolamine of 0.05 m , 600 l adenine of 90 mm , 600 l progesterone of 2 nm , 720 l cacl2 of 1 m , 600 l triiodothyronine of 10 nm and 6 ml newborn calf serum . 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 x10 cells / ml in skin reconstruct medium i. 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 x10 cells / ml in skin reconstruct medium i. remove washing medium from both inside and outside of each insert . add skin reconstruct medium i ( 1.5 ml to the inside and 10 ml to the outside of each insert ) . take 600 l cell suspension of keratinocytes and 600 l cell suspension of melanocytes or melanoma cells , mix well . dispense 200 l mixed cell suspension drop by drop to the inside of each insert . for dermal stem cell reconstruct , use 100 l of keratinocyte suspension only . 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 ) . 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 . harvest skin reconstruct at day 18 : aspirate media from the inside and the outside of inserts.remove inserts from tray with forceps.cut out the reconstruct ( including the polycarbonate filter ) by tracing a circle close to the edge with a scalpel blade.cut the reconstruct and the filter in half on a hard surface . cut out the reconstruct ( including the polycarbonate filter ) by tracing a circle close to the edge with a scalpel blade . for paraffin sections : place a half of the reconstruct in a histology cassette between 2 black tbs biopsy papers and soak the whole cassette in 10% formalin for more than 4 hours . then place the cassette in 70% ethanol and store it at 4 c until you are ready to process the reconstruct for paraffin embedding . for frozen sections : place the other half of reconstruct in 50% sucrose at 4 c for 1 - 2 hours , then change the sucrose to 2 m and store it at 4 c for another 1 - 2 hours.dispense optimal cutting temperature freezing media ( oct ) into a disposable base mold so that it fills about 50% of the boat . avoid any bubbles in the oct.grab the edge of the reconstruct with forceps and remove the reconstruct from the sucrose . place it on kimwipes until the kimwipes absorb the sucrose.transfer the reconstruct into the base mold on top of the oct , using forceps and a spatula . cover the top of the reconstruct with more oct until the base mold is completely full . make sure you do not have any bubbles in the oct which will make cutting difficult.place the base mold evenly on crushed dry ice , and allow the oct to freeze completely.wrap the base mold in tin foil and store it at -70 c until you are ready to cut it with a cryostat . place the other half of reconstruct in 50% sucrose at 4 c for 1 - 2 hours , then change the sucrose to 2 m and store it at 4 c for another 1 - 2 hours . dispense optimal cutting temperature freezing media ( oct ) into a disposable base mold so that it fills about 50% of the boat . grab the edge of the reconstruct with forceps and remove the reconstruct from the sucrose . transfer the reconstruct into the base mold on top of the oct , using forceps and a spatula . cover the top of the reconstruct with more oct until the base mold is completely full . make sure you do not have any bubbles in the oct which will make cutting difficult . place the base mold evenly on crushed dry ice , and allow the oct to freeze completely . wrap the base mold in tin foil and store it at -70 c until you are ready to cut it with a cryostat . graft a skin reconstruct on a mouse : prepare a 50 ml falcon tube with 5 ml of dmem ( for freezing and defrosting mouse skin).use a female scid hairless outbred mouse around 6 weeks.the mouse is anesthetized with isoflurane ( ez anesthesia system ) : initial anesthesia is performed in the induction chamber with 4% isoflurane ( before mouse is moved to the surgical bed ) and maintain anesthesia through a nosecone breather during procedure ( isoflurane : 1.5 - 2% ) . heat support by a heated pad to prevent hypothermia and sterile ophthalmic lubricant applied to prevent ocular injury during anesthesia.put 600 ml water in a beaker and leave on top of a hot plate to keep water temperature between 40 - 60 c.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 suturing later.cut a round wound bed about 1.2 cm in diameter on the mouse upper back using iris scissors and forceps . cover the wound bed with sterile gauze sponges . put the round mouse skin in 5 ml of dmem , then leave in liquid nitrogen container until it totally frozen . defrost the tube in hot water on top of a hot plate until totally thawed . repeat 3 times.detach the skin reconstruct from the transwell using surgical blade and remove the membrane very carefully , transfer the skin reconstruct ( epidermis side up ) on top of the wound bed.place the mouse skin ( epidermis side up ) on top of the skin reconstruct.make the first suture on the marker previously labeled , the second on the bottom , then two more sutures on sides to fix the mouse skin.clean mouse skin after grafting . remove nosecone and keep mouse on the heated pad until awake and ambulatory.put the mouse into a new cage.post operative analgesia : meloxicam ( 1 mg / kg ) by subcutaneous injection immediately after surgery , 24 hours and 48 hours after surgery . prepare a 50 ml falcon tube with 5 ml of dmem ( for freezing and defrosting mouse skin ) . use a female scid hairless outbred mouse around 6 weeks . the mouse is anesthetized with isoflurane ( ez anesthesia system ) : initial anesthesia is performed in the induction chamber with 4% isoflurane ( before mouse is moved to the surgical bed ) and maintain anesthesia through a nosecone breather during procedure ( isoflurane : 1.5 - 2% ) . heat support by a heated pad to prevent hypothermia and sterile ophthalmic lubricant applied to prevent ocular injury during anesthesia . put 600 ml water in a beaker and leave on top of a hot plate to keep water temperature between 40 - 60 c . 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 suturing later . cut a round wound bed about 1.2 cm in diameter on the mouse upper back using iris scissors and forceps . put the round mouse skin in 5 ml of dmem , then leave in liquid nitrogen container until it totally frozen . defrost the tube in hot water on top of a hot plate until totally thawed . detach the skin reconstruct from the transwell using surgical blade and remove the membrane very carefully , transfer the skin reconstruct ( epidermis side up ) on top of the wound bed . place the mouse skin ( epidermis side up ) on top of the skin reconstruct . make the first suture on the marker previously labeled , the second on the bottom , then two more sutures on sides to fix the mouse skin post operative analgesia : meloxicam ( 1 mg / kg ) by subcutaneous injection immediately after surgery , 24 hours and 48 hours after surgery . skin reconstructs with normal melanocytes and dermal stem cells the skin reconstructs are cultured in a special 6-well tray with inserts . the dermal compartment is cultured in dmem with 10% fbs for the first four days ( fig . the skin reconstruct medium i is used when keratinocytes are seeded with melanocytes or melanoma cells ( fig . the epidermis is exposed to the air at day 9 and this allows keratinocytes to differentiate ( fig . 1c ) . at day 18 , the epidermis of skin reconstructs is composed of stratified keratinocyte layers . staining the section of reconstructs with the melanocytic marker s100 shows that melanocytes are aligned in the basal layer of the epidermis and communicate with multiple keratinocytes through dendrite extensions ( fig . deposited collagen iv indicates the basement membrane , which separates the epidermis from the dermis ( fig . when dermal stem cells ( labeled with gfp lentiviral vector ) are embedded with fibroblasts in a collagen i matrix , they migrate to the epidermis and differentiate into melanocytes ( 1 ) , ( fig . skin reconstructs of melanoma tumors clinicopathological studies indicate that melanomas progress in a stepwise manner : common acquired nevi , dysplastic nevi , rgp ( radial growth phase ) melanomas , vgp ( vertical growth phase ) melanomas and metastatic melanomas ( 7 ) . different stages of melanoma cell lines are morphologically similar to each other in 2-d culture ( fig . 3a - d ) but when they are incorporated in skin reconstructs , the behavior of the cells reflects their in vivo characteristics . the location and the growth rate of normal melanocytes are tightly controlled in skin reconstructs ( fig . a. fibroblasts mixed with collagen are grown in dmem containing 10% fbs and form dermal compartment . b. keratinocytes and melanocytes are seeded on top of the dermis and grow in skin reconstruct medium . c. the epidermis is exposed to air at day 9 . d. h&e - stained skin reconstruct presents the epidermis comprised vertically , oriented basal layer , and sequentially differentiated stratified cell layers . e. s100-positive melanocytes ( black arrows ) are aligned at the basement membrane and communicate with multiple keratinocytes . f. collagen iv - staining indicates the basement membrane , which separates the epidermis from the dermis . all the stainings in d - f were performed on formalin - fixed , paraffin - embedded sections . dermal stem cells in skin reconstructs migrate to the epidermis and differentiate into melanocytes . at day 5 after seeding keratinocytes , single gfp - positive cells ( green ) the epidermis is still composed of a single layer . at day 8 , a few cells reach the epidermis - dermis interface . by day 10 the migrated gfp - positive cells in the epidermis express melanocytic marker hmb45 ( red , as indicated by white arrows ) . g. vgp melanoma wm793 cells invade into the dermis through basement membrane . h. metastatic melanoma 1205lu cells aggressively invade deep into dermis . we have described generating 3d skin reconstructs with normal human melanocytes , dermal stem cells and melanoma cells . when grown in monolayer culture , melanocytic cells present a similar morphology ( flattened , spindle or more dendritic - shaped ) regardless of their origin of clinical stage . in contrast normal human melanocytes reside at the basement membranes between the epidermal and the dermal layers as single cells . rgp primary melanoma cells grow as small clusters along the basement membrane whereas more aggressive vgp melanoma cells grow as large clusters breaking through the basement membrane into the dermis . metastatic melanoma cells grow in all directions and invade deep into the dermis as single cells or clusters . quantitative analyses can be performed on this model by measuring the depth of invasion and the extent of proliferation . in addition to characterization of normal and malignant melanocytic cells , using the model we can directly induce the differentiation of dermal stem cells into matured melanocytes , which home to the basal layer of epidermis and establish bona fide communication with keratinocytes through upregulation of e - cadherin ( 6 ) .   using viral vectors , we are able to either activate or inactivate gene function for a better understanding of the dynamics and functional significance of genes expressed by each cell type in skin reconstructs , which promises to be an efficient model to study not only transformation mechanisms of melanocytes , but also progression of melanoma ( 8) . long - term observation of cell phenotypes has become possible through grafting of skin reconstructs to immunodeficient animals . such a human skin - mouse chimera is an excellent research tool to study melanomagenesis and melanoma metastasis . many drugs which eradicate cancer cells in 2d culture conditions often have little effect in experimental and clinical applications . as seen in the in vivo tumor , melanoma cells in 3d cultures are often resistant to the drugs which cells in 2d cultures respond to , suggesting that the microenvironment modulates signaling pathways in melanoma . for successful drug discovery , the 3d skin reconstruct model is an ideal preclinical tool to predict the effects of compounds in vivo ( 9,10 ) .   in summary , they will lead to a better understanding of which genes are involved in transformation and how stem cells contribute to that transformation .
most in vitro studies in experimental skin biology have been done in 2-dimensional ( 2d ) monocultures , while accumulating evidence suggests that cells behave differently when they are grown within a 3d extra - cellular matrix and also interact with other cells ( 1 - 5 ) . 
 mouse models have been broadly utilized to study tissue morphogenesis in vivo . 
 however mouse and human skin have significant differences in cellular architecture and physiology , which makes it difficult to extrapolate mouse studies to humans . 
 since melanocytes in mouse skin are mostly localized in hair follicles , they have distinct biological properties from those of humans , which locate primarily at the basal layer of the epidermis . 
 the recent development of 3d human skin reconstruct models has enabled the field to investigate cell - matrix and cell - cell interactions between different cell types . 
 the reconstructs consist of a " dermis " with fibroblasts embedded in a collagen i matrix , an " epidermis " , which is comprised of stratified , differentiated keratinocytes and a functional basement membrane , which separates epidermis from dermis . 
 collagen provides scaffolding , nutrient delivery , and potential for cell - to - cell interaction . 
 the 3d skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin . as in vivo 
 , melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes . 
 melanoma cells exhibit the same characteristics reflecting the original tumor stage ( rgp , vgp and metastatic melanoma cells ) in vivo . 
 recently , dermal stem cells have been identified in the human dermis ( 6 ) . 
 these multi - potent stem cells can migrate to the epidermis and differentiate to melanocytes .