High-throughput, high-content screening for novel pigmentation regulators using a keratinocyte/melanocyte co-culture system

Exp Dermatol. 2014 Feb;23(2):125-9. doi: 10.1111/exd.12322.


Skin pigmentation is a complex process including melanogenesis within melanocytes and melanin transfer to the keratinocytes. To develop a comprehensive screening method for novel pigmentation regulators, we used immortalized melanocytes and keratinocytes in co-culture to screen large numbers of compounds. High-throughput screening plates were subjected to digital automated microscopy to quantify the pigmentation via brightfield microscopy. Compounds with pigment suppression were secondarily tested for their effects on expression of microphthalmia transcription factor (MITF) and several pigment regulatory genes, and further validated in terms of non-toxicity to keratinocytes/melanocytes and dose-dependent activity. The results demonstrate a high-throughput, high-content screening approach, which is applicable to the analysis of large chemical libraries using a co-culture system. We identified candidate pigmentation inhibitors from 4000 screened compounds including zoxazolamine, 3-methoxycatechol and alpha-mangostin, which were also shown to modulate expression of MITF and several key pigmentation factors and are worthy of further evaluation for potential translation to clinical use.

Keywords: co-culture; high-content screening; keratinocyte; melanocyte; microphthalmia transcription factor; pigmentation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line, Transformed
  • Coculture Techniques
  • Enzyme Inhibitors / isolation & purification*
  • Enzyme Inhibitors / pharmacology
  • Gene Expression Regulation / drug effects*
  • High-Throughput Screening Assays*
  • Intramolecular Oxidoreductases / biosynthesis
  • Intramolecular Oxidoreductases / genetics
  • Keratinocytes / drug effects*
  • Keratinocytes / metabolism
  • Melanins / biosynthesis*
  • Melanins / genetics
  • Melanocytes / drug effects*
  • Melanocytes / metabolism
  • Melanoma, Experimental / pathology
  • Membrane Glycoproteins / biosynthesis
  • Membrane Glycoproteins / genetics
  • Mice
  • Microphthalmia-Associated Transcription Factor / biosynthesis
  • Microphthalmia-Associated Transcription Factor / genetics
  • Monophenol Monooxygenase / biosynthesis
  • Monophenol Monooxygenase / genetics
  • Oxidoreductases / biosynthesis
  • Oxidoreductases / genetics
  • Skin Pigmentation / drug effects*


  • Enzyme Inhibitors
  • Melanins
  • Membrane Glycoproteins
  • Microphthalmia-Associated Transcription Factor
  • Mitf protein, mouse
  • Oxidoreductases
  • Tyrp1 protein, mouse
  • Monophenol Monooxygenase
  • Intramolecular Oxidoreductases
  • dopachrome isomerase