Melanocyte culture

Summary

After trypsin digestion, epidermal slices isolated from skin slices were dispersed with EDTA and then incubated with serum-free culture medium supplemented with bFGF (FGF-2).

Operation method

Program 23.21 Culture of melanocytes

Principle

After trypsin digestion, epidermal slices isolated from skin slices were dispersed with EDTA and then incubated with serum-free culture medium supplemented with bFGF (FGF-2).

Materials and Instruments

D-PBSA Fetal bovine serum Soybean trypsin inhibitor
Hormone-added melanocyte culture solution Trypsin and EDTA mixture
Tissue culture dishes Straws Centrifuge tubes Humidified incubators Water baths Electronic cell counters or blood cell counting plates

Move

Primary culture

Day 1

1. Wash the skin specimen with 70 % ethanol, then soak it twice with D-PBSA.

2. Transfer the tissue to a sterile 100 mm dish, epidermis side down.

3. Remove subcutaneous fat and deep dermis with dissecting scissors.

4. Cut the remaining tissue into 2 mm × 2 mm pieces with a scalpel. Swing the blade over the tissue, but do not use a saw cut.

5. Transfer the tissue pieces into a 15 ml centrifuge tube containing cold 0.25 % trypsin.

6. Incubate the blocks for 60-90 min at room temperature. For some donor skins, incubate at 4°C for 18-24 h and then at 37°C for 1-2 h to obtain a higher number of melanocytes.

Day 2

7. Tap the centrifuge tube to move the tissue mass that has settled to the bottom. Then, pour the contents of the centrifuge tube quickly into a 60 mm Petri dish.

8. Using surgical forceps, transfer the tissue blocks one by one into a 100 mm dry petri dish, epithelial side down. Gently shake the block so that it touches the bottom wall of the dish. The epidermis should be attached to the dish so that the dermis can be thoroughly separated with surgical forceps. Remove the dermal piece.

9. Transfer the epidermal slice into a 15 ml centrifuge tube containing 5 ml of 0.02% EDTA (0.7 mmol/L). Be careful to place the epidermal piece into the EDTA solution and not against the wall of the centrifuge tube.

10. Gently shake the tube to disperse the epidermal slices into single cells.

11. Centrifuge (350 g for 5 min) and aspirate the supernatant.

12. Suspend the cells with MHSM.

13. Count the cells with a hemocyte counter plate, and then incubate the cells in a 35 mm dish with 2 ml of MHSM containing 5 % FBS at a density of ^1×106 cells (about ^{2×104~4×104} melanocytes). FBS promotes cell attachment.

14. Change the solution with MHSM containing growth factors and bovine pituitary extract twice a week.

Day 3~30

15. After 24 h of incubation, the cultured cells contained primary keratinocytes and scattered melanocytes. Keratinocytes stopped proliferating after a few days, and keratinocyte clones surfaced in week 2. At the end of week 2, only melanocytes remained. In most cases, the cells grow close to confluence within 2 to 4 weeks, at which point they are ready for passaging.



Passage Culture

16. Gently wash the cells 2 times with 0.02% EDTA (0.7 mmol/L).

17. Add 3 ml of a mixture of 0.25 % trypsin and 0.1 % EDTA (2.5 mmol/L) and incubate at 37°C. Every 5 min, the cells were visualized under a phase contrast microscope. The cells were observed under a phase contrast microscope every 5 min to check for de-adhesion.

18. When most of the cells are de-attached, trypsin is inactivated with 10 μg/ml soybean trypsin inhibitor or 1 ml of culture medium containing calf serum. During passaging, "haemostimulation" is useful to maintain the growth of NSCs under serum-free conditions.

19. Blow the cells in the culture dish to ensure that all melanocytes are de-attached.

20. Aspirate the cell suspension and centrifuge (350 g for 5 min).

21. Aspirate the supernatant and suspend the cells with Calcium Free Melanocyte Culture Solution containing 5 % FBS. ^{2×104~4×104} cells were placed in each 100 mm dish. The melanocytes adhere well to the plastic dishes (> 75 %) with FBS, but FBS may not be used if the dishes are coated with fibronectin or a mixture of collagen type I and collagen type III [Gilchres te al., 1985].

22. Change the culture medium of melanocytes with serum-free or blood-collecting medium twice a week. Cultures with serum-containing medium yield more cells, but culture medium with haematocrit promotes fibroblast growth. Reducing the ^Ca2+ concentration to 0.03 mmd/L did not affect melanocyte proliferation but inhibited fibroblast overgrowth [Naeyaert et al., 1991].

Melanocyte hormone-supplemented medium (MHSM):

(a) 199 culture medium (400-1200, GIBCO): 93.1 ml;

(b) EGF (B-D Biosciences): 0.1 ml at a storage concentration of 10 μg/ml and a final concentration of 10 ng/ml;

(c) Transferrin (Sigma): 0.1 ml at a storage concentration of 10 mg/ml and a final concentration of 10 μg/ml;

(d) Insulin (Sigma): 0.1 ml, stored at a concentration of 10 mg/ml and a final concentration of 10 μg/ml;

(e) Triiodothyronine (Sigma): 0.1 ml, stored at a concentration of 1 μmol/L, final concentration of 1 nmol/L;

(f) Cortisone oxide (Calbiochem): 0.5 ml, stored at a concentration of 0.28 mmol/L, final concentration of 1.4 μmol/L;

(g) Cholera toxin (Calbiochem): 1.0 ml at a storage concentration of 1 μmol/L and a final concentration of 1 nmol/L. Bovine pituitary extract and cyclobenzaprine dibutyrate may be used in place of cholera toxin. Bovine pituitary extract (Clcmetics) has a storage concentration of 35 μg/ml and a final concentration of 0.7 ng/ml. cyclobenzaprine dibutyrate (Sigma) has a storage concentration of 1 mmol/L and a final concentration of 100 μmol/L;

(h) bFGF (Amgen): 5.0 ml at a storage concentration of 200 ng/ml and a final concentration of 10 ng/ml.

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