Improvement of Cell Culture Methods for the Successful Generation of Human Keratinocyte Primary Cell Cultures Using EGF-Loaded Nanostructured Lipid Carriers Chato Astrain, Jesús Sánchez Porras, David García García, Óscar Darío Vairo, Claudia Villar Vidal, María Villullas, Silvia Sánchez-Montesinos García, Indalecio Campos Sánchez, Fernando Garzón Bello, Ingrid Johanna Alaminos Mingorance, Miguel EGF Nanoparticles NLC Keratinocytes Tissue engineering Cell culture 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 explantbased methods in the efficient generation of human keratinocyte primary cell cultures for tissue engineering use. 2021-11-08T11:32:13Z 2021-11-08T11:32:13Z 2021 info:eu-repo/semantics/article Chato-Astrain, J.; SánchezPorras, D.; García-García, Ó.D.; Vairo, C.; Villar-Vidal, M.; Villullas, S.; Sánchez-Montesinos, I.; Campos, F.; Garzón, I.; Alaminos, M. Improvement of Cell Culture Methods for the Successful Generation of Human Keratinocyte Primary Cell Cultures Using EGF-Loaded Nanostructured Lipid Carriers. Biomedicines 2021, 9, 1634. https://doi.org/10.3390/ biomedicines9111634 http://hdl.handle.net/10481/71365 10.3390/biomedicines9111634 eng http://creativecommons.org/licenses/by/3.0/es/ info:eu-repo/semantics/openAccess Atribución 3.0 España MDPI