Biomedicines (Nov 2021)

Improvement of Cell Culture Methods for the Successful Generation of Human Keratinocyte Primary Cell Cultures Using EGF-Loaded Nanostructured Lipid Carriers

  • Jesús Chato-Astrain,
  • David Sánchez-Porras,
  • Óscar Darío García-García,
  • Claudia Vairo,
  • María Villar-Vidal,
  • Silvia Villullas,
  • Indalecio Sánchez-Montesinos,
  • Fernando Campos,
  • Ingrid Garzón,
  • Miguel Alaminos

DOI
https://doi.org/10.3390/biomedicines9111634
Journal volume & issue
Vol. 9, no. 11
p. 1634

Abstract

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Human skin keratinocyte primary cultures can be established from skin biopsies with culture media containing epithelial growth factor (EGF). Although current methods are efficient, optimization is required to accelerate the procedure and obtain these cultures in less time. In the present study, we evaluated the effect of novel formulations based on EGF-loaded nanostructured lipid carriers (NLC). First, biosafety of NLC containing recombinant human EGF (NLC-rhEGF) was verified in immortalized skin keratinocytes and cornea epithelial cells, and in two epithelial cancer cell lines, by quantifying free DNA released to the culture medium. Then we established primary cell cultures of human skin keratinocytes with basal culture media (BM) and BM supplemented with NLC-rhEGF, liquid EGF (L-rhEGF), or NLC alone (NLC-blank). The results showed that cells isolated by enzymatic digestion and cultured with or without a feeder layer had a similar growth rate regardless of the medium used. However, the explant technique showed higher efficiency when NLC-rhEGF culture medium was used, compared to BM, L-rhEGF, or NLC-blank. Gene expression analysis showed that NLC-rhEGF was able to increase EGFR gene expression, along with that of other genes related to cytokeratins, cell–cell junctions, and keratinocyte maturation and differentiation. In summary, these results support the use of NLC-rhEGF to improve the efficiency of explant-based methods in the efficient generation of human keratinocyte primary cell cultures for tissue engineering use.

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