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Melanocyte Growth and Isolation

Day 1

1. Prepare the following in a laminar flow hood: one pair of sterile forceps, curved scissors, and surgical scalpel blade; 5 ml of dispase II (Boehringer Mannheim) in a sterile centrifuge tube; 20 ml of Ca2+, Mg2+ -free HBSS in a sterile non-tissue culture Petri dish; and 10 ml of 70% ethanol in a separate Petri dish.

2. Soak the skin specimens in 70% ethanol for 30 seconds. Transfer skin to another Petri dish containing HBSS to rinse off ethanol (see notes 1 and 2).

3. Cut skin-ring open and trim off fat and subcutaneous tissue with scissors (see note 3).

4. Cut skin into pieces (approximately 5 x 5 mm2) using the surgical scalpel blade with one-motion cuts (see note 4).

5. Transfer the pieces into the tube containing dispase II. Cap, invert, and incubate the tube in the refrigerator at 4°C for 15-18 hours.

Day 2

1. Prepare the following in a laminar flow hood: one pair of sterile forceps and a surgical scalpel blade; 10 ml of Ca2+, Mg2+ -free HBSS in a sterile, non-tissue culture Petri dish; two empty sterile Petri dishes; 2 ml of 0.5% trypsin/versene solution in a 15-ml centrifuge tube; and 8 ml of soybean trypsin inhibitor.

2. Pour tissues in dispase II into one of the empty Petri dishes. Transfer tissue pieces to the Petri dish. Separate epidermis (thin translucent layer) from dermis (thick opaque layer) using the forceps and scalpel blade. Hold the dermal part of the skin piece with the forceps, and gently slide the epidermal side off with a scalpel blade. Discard the dermis immediately (see note 5).

3. Add a drop of Ca2+, Mg2+ -free HBSS on the resulting epidermis to avoid drying while isolating the epidermis from the remaining skin pieces. Repeat the procedure in step 2 or each piece of tissue. Mince the epidermal sheets as small as possible using a surgical scalpel blade.

4. Transfer the collected epidermis from the Petri dish to a centrifuge tube containing 0.5% trypsin/versene solution. Incubate the tube at 37°C for 3-5 minutes depending on cell disaggregation. Pipette up and down vigorously 80x with a 5 ml pipette to release single cells from epidermal sheets. Neutralize trypsin with soybean trypsin inhibitor. Centrifuge for 5 minutes at 1,200 rpm at room temperature. Aspirate the supernatant which may contain remaining stratum corneum. Resuspend the pellet with Melanoblast Media.

5. Place the resulting epidermal cell suspension at approximately concentration 2 x 105 cells/cm2 in the tissue culture vessel. Incubate at 37°C 5% CO2 for 48-72 hours.

After 2 Days

1. Change media to new Melanoblast Media to remove non-adherent cells. Medium change should be performed twice each week. If the culture is contaminated with fibroblasts, start treatment with MGM containing 100 ug/ml [ID1] of geneticin (G418, Invitrogen Cat. No. 11811) for 2-3 days. Seventy percent confluent primary cultures can be obtained in 2 weeks.

2. Subcultivation. Primary cultures established from foreskins usually reach 70% confluence within 12 days. Cultures are washed with Ca2+, Mg2+ -free HBSS and treated with trypsin-versene solution (90 ml versene + D-glucose and 10 ml 2.5% trypsin) at room temperature for 1 minute, harvested with Leibovitz's L-15 containing 10% heat-inactivated FCS, centrifuged at 1,200 rpm for 5 minutes, resuspended in Melanoblast Media, reinoculated at 104 cells/cm2, and serially passaged. Medium is changed twice each week.

3. Cryopreservation. Melanocyte suspensions harvested by trypsin-versene and Leibovitz's L-15 containing 10% FCS are centrifuged at 1,200 rpm for 5 minutes and resuspended in heat-inactivated FCS containing 5% DMSO as a cryopreservative. Cells are normally suspended at a density of 106 /ml and transferred to cryotubes. The tubes are then placed in a plastic sandwich box (NalgeneTM Cryo 1°C Freezing Container, Cat. No. 5100-00001) which is immediately transferred to a -70°C freezer. The insulation of the box ensures gradual cooling of the cryotubes and results in over 80% viability of the cells upon thawing. After overnight storage in the freezer, the cryotubes are placed in permanent storage in liquid nitrogen.

4. Thawing. The melanocyte suspension is thawed by placing the cryotube in a water bath at 37°C. When the cell-preservative medium is almost but not totally defrosted, the outside of the tube is wiped with 70% ethanol. The cell suspension is then withdrawn, quickly diluted in Leibovitz's L-15 containing 10% FCS at room temperature, centrifuged, and resuspended in fresh Melanoblast Media. Cell viability is determined by trypan blue exclusion. The resulting melanocytes are then seeded at a density of 104/cm2.

  

NOTES:

1. Tissue Source and Collection: The sources of tissue for melanocyte cultures are human neonatal foreskins obtained from routine circumcision and normal adult skin acquired from reduction mammoplasty. At the time of excision, the skin is placed into a sterile container with 20 ml of normal skin transporting medium (2 ml penicillin/streptomycin + 2 ml gentamycin in 1L Ca2+, Mg2+ -free HBSS) supplied in advance and kept near the surgical area at 4°C. Specimens are delivered immediately to the tissue culture laboratory or stored at 4°C. Neonatal foreskins can be kept for up to 48 hours and normal adult skin, for up to 24 hours. However, the fresher the specimens, the higher the yield of live cells upon isolation.

2. Sterilization of Skin Specimens: Reduce contamination by a short treatment (30 seconds) of intact skin with 70% ethanol in a laminar flow hood. After sterilization, rinse samples with HBSS.

3. Preliminary Tissue Preparation: Place tissues in a 100-mm non-tissue culture Petri dish and remove most of the subcutaneous fat and membranous material with curved scissors.

4. Adjustment of Tissue Size for Trypsinization: To improve reagent penetration, cut the skin samples into small pieces (approximately 5 x 5 mm2) rinsed in HBSS.

5. Separation of Epidermis from Dermis: Dispase II splits epidermis from the dermis along the basement membrane. Since melanocytes are located just above the basement membrane, removal of this lowest layer of epidermal cells requires some effort. Each piece of skin is held with forceps with dermal side down. The epidermal sheet is detached by sliding it off with a scalpel blade. To prevent the epidermis from drying, a drop of Ca2+, Mg2+ -free HBSS can be added to the resulting epidermal sheet. To avoid potential sources of fibroblast contamination, dermal pieces should be discarded immediately once they are separated from the epidermis, and the forceps used to hold the dermis should never touch the epidermal sheets. Contaminated dermis is easily recognized by its white color in contrast to the yellowish-brown color of the epidermis. Isolated epidermal sheets in cell dispersal solution are then transferred to a centrifuge tube.