Isolation, Culture, and Transfection of Melanocytes

Lauren S. Godwin1, Joanna T. Castle1, Jaskaren S. Kohli1, Philip S. Goff1, Claire J. Cairney2, W. Nicol Keith2, Elena V. Sviderskaya1, Dorothy C. Bennett1

1 Molecular Cell Biology Group, Biomedical Sciences Research Centre, St. George's, University of London, London, 2 Institute of Cancer Sciences, University of Glasgow, Glasgow, Scotland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 1.8
DOI:  10.1002/0471143030.cb0108s63
Online Posting Date:  June, 2014
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Abstract

ABSTRACT

Located in the basal epidermis and hair follicles, melanocytes of the integument are responsible for its coloration through production of melanin pigments. Melanin is produced in lysosomal‐like organelles called melanosomes. In humans, this skin pigmentation acts as an ultraviolet radiation filter. Abnormalities in the division of melanocytes are quite common, with potentially oncogenic growth usually followed by cell senescence producing benign naevi (moles), or occasionally melanoma. Therefore, melanocytes are a useful model for studying melanoma, as well as pigmentation and organelle transport and the diseases affecting these mechanisms. This chapter focuses on the isolation, culture, and transfection of human and murine melanocytes. The first basic protocol describes the primary culture of melanocytes from human skin and the maintenance of growing cultures. The second basic protocol details the subculture and preparation of mouse keratinocyte feeder cells. The primary culture of melanocytes from mouse skin is described in the third basic protocol, and, lastly, the fourth basic protocol outlines a technique for transfecting melanocytes and melanoma cells. Curr. Protoc. Cell Biol. 63:1.8.1‐1.8.20. © 2014 by John Wiley & Sons, Inc.

Keywords: melanocytes; primary culture; epidermis; keratinocytes; human; mouse

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Primary Explantation of Human Melanocytic Cells
  • Basic Protocol 2: Preparation of Keratinocyte Feeder Cells for Use in the Primary Culture of Mouse Melanocytes
  • Basic Protocol 3: Primary Culture of Melanocytes from Mouse Skin
  • Basic Protocol 4: Transfection of Human Melanocytes and Melanoma Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Primary Explantation of Human Melanocytic Cells

  Materials
  • Iodine solution (see recipe)
  • 70% ethanol
  • Dulbecco's phosphate‐buffered saline (DPBS) without calcium or magnesium (e.g., Invitrogen, cat. no. 14190‐094)
  • 10 µg/ml gentamicin in DPBS without Ca or Mg
  • Skin sample (may be surplus postoperative skin or obtained by punch biopsy—at least 5 mm diameter; best results are obtained with at least 1 cm2 of skin)
  • 10 mg/ml dispase II in Hank's balanced salt solution
  • 500 µg/ml trypsin/EDTA solution (see recipe)
  • RPMI growth medium (see recipe)
  • Mitogens:
    • 40 µM TPA (12‐O‐tetradecanoylphorbol 13‐acetate) (see recipe)
    • 40 nM CT (cholera toxin) (see recipe)
    • 5 µM ET1 (endothelin 1) (see recipe)
    • 5 µg/ml SCF (human stem cell factor) (see recipe)
  • 125 µg/ml trypsin/EDTA solution (see recipe)
  • Dimethylsulfoxide (DMSO)
  • Liquid nitrogen
  • Phenylthiourea (PTU; see recipe)
  • 6‐well plates
  • Surgical equipment including:
    • Forceps
    • Surgical scalpels
  • 2.5‐ml Combitips and dispenser
  • Hemacytometer (unit 1.1)
  • 2‐ml cryotubes
  • 30‐ml universal tube (e.g., Elkay Laboratory Products, cat. no. 500‐1000‐302)
  • Additional reagents and equipment for counting cells using a hemacytometer (unit 1.1)

Basic Protocol 2: Preparation of Keratinocyte Feeder Cells for Use in the Primary Culture of Mouse Melanocytes

  Materials
  • XB2 mouse keratinocytes (available from the Wellcome Trust Functional Genomics Cell Bank; see Internet Resources) growing in culture
  • Dulbecco's phosphate‐buffered saline (DPBS) without calcium or magnesium (e.g., Invitrogen, cat. no. 14190‐235)
  • Disodium EDTA
  • 250 µg/ml trypsin/EDTA solution (see recipe)
  • DMEM growth medium (see recipe)
  • Mitomycin C stock (see recipe)
  • 30‐ml universal tubes (e.g., Elkay Laboratory Products, cat. no. 500‐1000‐302)
  • Culture flasks
  • Hemacytometer (unit 1.1)
  • Additional reagents and equipment for counting cells using a hemacytometer (unit 1.1) and freezing cells ( protocol 1, step 18)

Basic Protocol 3: Primary Culture of Melanocytes from Mouse Skin

  Materials
  • Keratinocyte feeder cells ( protocol 2)
  • RPMI growth medium (see recipe)
  • Mouse pups (up to 3 days old) or pregnant mouse
  • 70% ethanol
  • Dulbecco's phosphate‐buffered saline (DPBS) without calcium or magnesium (e.g., Invitrogen, cat. no. 14190‐235)
  • 5 mg/ml trypsin (see recipe)
  • 250 µg/ml trypsin/EDTA solution (see recipe)
  • 1 mg/ml soybean trypsin inhibitor (see recipe)
  • 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) (see recipe)
  • Cholera toxin (see recipe)
  • 35‐mm culture dishes
  • 50‐mm (4‐ml) and 9‐cm petri dishes
  • Dissecting equipment including two pairs of fine forceps and two curved scalpels
  • Dissecting microscope in culture hood (need sterile atmosphere)
  • 30‐ml universal tubes
  • 2.5 ml Combitips
  • Culture flasks
  • Additional reagents and equipment for dissection of pregnant mouse to obtain embryos (e.g., unit 19.13), propagating melanocyte cultures ( protocol 1, steps 12 to 17), counting cells (unit 1.1), and freezing and thawing cells ( protocol 1, step 18)

Basic Protocol 4: Transfection of Human Melanocytes and Melanoma Cells

  Materials
  • siRNAs:
    • Negative control siRNA—Silencer Select negative control (siRNA #2; Ambion, cat. no. 4390846)
    • Positive control siRNA—CDC2 siRNA gene solution (Qiagen, cat. no. 1027416 gene ID 983)
    • Transfection positive control siRNA—AllStars Hs Cell Death Control siRNA (Qiagen, cat. no. 1027298)
    • Test siRNAs—Silencer Select validated or predesigned (inventoried) siRNA Life Technologies (Ambion)
  • RNase‐free distilled H 2O
  • RPMI 1640 medium
  • Fetal bovine serum
  • L‐glutamine
  • Mitogens:
    • 40 µM TPA (12‐O‐tetradecanoylphorbol 13‐acetate) (see recipe)
    • 40 nM CT (cholera toxin) (see recipe)
    • 5 µM ET1 (endothelin 1) (see recipe)
    • 5 µg/ml SCF (human stem cell factor) (see recipe)
  • 125 µg/ml trypsin/EDTA solution (see recipe)
  • Lipofectamine 2000 (Life Technologies)
  • Human melanocyte or melanoma cell suspension ( protocol 1)
  • Nunc 96‐well black optical‐bottom tissue culture plates
  • Multichannel pipettors and reservoirs
  • Additional reagents and equipment for trypsinization ( protocol 1)
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Figures

Videos

Literature Cited

  Bennett, D.C. 2008. How to make a melanoma: What do we know of the primary clonal events? Pigment Cell Melanoma Res. 21:27‐38.
  Bennett, D.C., Cooper, P.J., and Hart I.R. 1987. A line of non‐tumorigenic mouse melanocytes, syngeneic with the B16 melanoma and requiring a tumour promoter for growth. Int. J. Cancer 39:414‐418.
  Bruno, A., Bruno, L., and Levi, V. 2013. Extracting the stepping dynamics of molecular motors in living cells from trajectories of single particles. Cell Biochem. Biophys. 65:1‐11.
  Costin, G. and Hearing, V.J. 2007. Human skin pigmentation: Melanocytes modulate skin colour in response to stress. Fed. Am. Soc. Exp. Biol. 21:976‐994.
  Dykxhoorn, D.M., Novina, C.D., and Sharp, P.A. 2003. Killing the messenger: Short RNAs that silence gene expression. Nat. Rev. Mol. Cell Biol. 4:457‐467.
  Eisinger, M. and Marko, O. 1982. Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin. Proc. Natl. Acad. Sci. U.S.A. 79:2018‐2022.
  Hachiya, A., Kobayashi, A., Yoshida, Y., Kitahara, T., Takema, Y., and Imokawa, G. 2004. Biphasic expression of two paracrine melanogenic cytokines, stem cell factor and endothelin‐1, in ultraviolet B‐induced human melanogenesis. Am. J. Pathol. 165:2099‐2109.
  Hirobe, T., Shinpo, T., Higuchi, K., and Sano, T. 2010. Life cycle of human melanocytes is regulated by endothelin‐1 and stem cell factor in synergy with cyclic AMP and basic fibroblast growth factor. J. Dermatol. Sci. 57:123‐131.
  Hume, A.N. and Seabra, M.C. 2011. Melanosomes on the move: A model to understand organelle dynamics. Biochem. Soc. Trans. 39:1191‐1196.
  Kitano, Y. and Okada, N. 1983. Separation of the epidermal sheet by dispase. Br. J. Dermatol. 108:555‐560.
  Kobayashi, N., Nakagawa, A., Muramatsu, T., Yamashina, Y., Shirai, T., Hashimoto, M., Ishigaki, Y., Ohnishi, T., and Mori, T. 1998. Supranuclear melanin caps reduce ultraviolet induced DNA photoproducts in human epidermis. J. Invest. Dermatol. 1998:806‐810.
  Kunisada, T., Yoshida, H., Ogawa, M., Shultz, L.D., and Nishikawa, S. 1996. Characterization and isolation of melanocyte progenitors from mouse embryos. Dev. Growth Different. 38:87‐97.
  Lamoreux, M.L., Delmas, V., Larue, L., and Bennett, D.C. 2010. The Colors of Mice: A Model Genetic Network. Wiley‐Blackwell, West Sussex, U.K.
  Lin, J. and Fisher, D. 2007. Melanocyte biology and skin pigmentation. Nature 445:843‐850.
  Mayer, T.C. 1973. The migratory pathway of neural crest cells into the skin of mouse embryos. Dev. Biol. 34:39‐46.
  Michaloglou, C., Vredeveld, L., Soengas, M., Denoyelle, C., Kuilman, T., van der Horst, C., Majoor, D., Shay, J., Mooi, W., and Peeper, D. 2005. BRAF E600‐associated senescence‐like cell cycle arrest of human naevi. Nature 436:720‐724.
  O'Keefe, E. and Cuatrecasas, P. 1974. Cholera toxin mimics melanocyte stimulating hormone in inducing differentiation in melanoma cells. Proc. Natl. Acad. Sci. U.S.A. 71:2500‐2504.
  Rheinwald, J.G. and Green, H. 1975. Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma. Cell 6:317‐330.
  Soo, J.K., Mackenzie Ross, A.D., Kallenberg, D.M., Milagre, C., Heung Chong, W., Chow, J., Hill, L., Hoare, S., Collinson, R.S., Hossain, M., Keith, W.N., Marais, R., and Bennett, D.C. 2011. Malignancy without immortality? Cellular immortalization as a possible late event in melanoma progression. Pigment Cell Melanoma Res. 24:490‐503.
  Stenn, K.S., Link, R., Moellmann, G., Madri, J., and Kuklinska, E. 1989. Dispase, a neutral protease from Bacillus polymyxa, is a powerful fibronectinase and type IV collagenase. J. Invest. Dermatol. 93:287‐290.
  Sviderskaya, E.V., Bennett, D.C., Ho, L., Bailin, T., Lee, S.T., and Spritz, R.A. 1997. Complementation of hypopigmentation in p‐mutant (pink‐eyed dilution) mouse melanocytes by normal human P cDNA, and defective complementation by OCA2 mutant sequences. J. Invest. Dermatol. 108:30‐34.
  Sviderskaya, E.V., Hill, S.P., Evans‐Whipp, T.J., Chin, L., Orlow, S.J., Easty, D.J., Cheong, S.C., Beach, D., DePinho, R.A., and Bennett, D.C.. 2002. p16(Ink4a) in melanocyte senescence and differentiation. J . Natl. Cancer Inst. 94:446‐454.
  Sviderskaya, E.V., Gray‐Schopfer, V.C., Hill, S.P., Smit, N.P., Evans‐Whipp, T.J., Bond, J., Hill, L., Bataille, V., Peters, G., Kipling, D., Wynford‐Thomas, D., and Bennett, D.C. 2003. P16/cyclin‐dependent kinase inhibitor 2A deficiency in human melanocyte senescence, apoptosis and immortalisation: Possible implications for melanoma progression. J. Natl. Cancer Inst. 95:723‐732.
  Sviderskaya, E.V., Kallenberg, D.M., and Bennett, D.C. 2010. Resource. The Wellcome Trust Functional Genomics Cell Bank: Holdings. Pigment Cell Melanoma Res. 23:147‐150.
  Tomasz, M. 1995. Mitomycin C: Small, fast and deadly (but very selective). Chem. Biol. 2:575‐579.
  Yamasaki, T., Takahashi, A., Pan, J., Yamaguchi, N., and Yokoyama, K.K. 2009. Phosphorylation of activation transcription factor‐2 at serine 121 by protein kinase C controls c‐Jun‐mediated activation of transcription. J. Biol. Chem. 284:8567‐8581.
Key References
  Bennett, et al., 1987. See above.
  The development of the first non‐tumorigenic melanocyte mouse line.
  Eisinger, and Marko, 1982. See above.
  A description of the first long‐term culture of human melanocytes.
  Sviderskaya et al., 1997. See above.
  Includes the current mouse melanocyte culture method.
Internet Resources
  http://www.sgul.ac.uk/depts/anatomy/pages/dcbm&m.htm
  Our materials and methods page with further and related details for melanocyte culture methods.
  http://www.sgul.ac.uk/depts/anatomy/pages/WTFGCB.htm
  Web site of Wellcome Trust Functional Genomics Cell Bank with access to among others mouse melanocyte and melanoblast lines carrying a variety of pigmentary mutations.
  http://www.espcr.org/micemut/
  Information on the mapped color genes in mice, zebrafish, and human models.
  http://genome.wellcome.ac.uk/doc_WTD020804.html
  An introduction to the mouse model organism.
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