Improvement of Cell Culture Methods for the Successful Generation of Human Keratinocyte Primary Cell Cultures Using EGF-Loaded Nanostructured Lipid Carriers
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AuthorChato 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
EGFNanoparticlesNLCKeratinocytesTissue engineeringCell culture
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
SponsorshipThis study was supported by the NanoGSkin project of EuroNanoMed-III (ERA-NET Cofund action of the Horizon 2020 Research and Innovation Framework Programme, EU), and cofunded by the Instituto de Salud Carlos III (ISCIII) through AES 2017 (ref. AC17/00013) and the Centro para el Desarrollo Tecnológico Industrial (CDTI) (ref. 00108589), Spanish Ministry of Science and Innovation. Support was also provided through projects PE-0395-2019 from the Consejería de Salud y Familias, Junta de Andalucía, Spain, and B-CTS-450-UGR20 (proyectos de I+D+i en el marco del Programa Operativo FEDER Andalucía 2014-2020, University of Granada and Consejería de Transformación Económica, Industria, Conocimiento y Universidades). Additional support was from the Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+i) of the Spanish Ministry of Science and Innovation through grants FIS PI20/0317 (ISCIII), FIS PI21/0980 (ISCIII) and RTC-2017-6696-1 (Nanogrow), co-financed by the European Regional Development Fund (ERDF) through the “Una manera de hacer Europa” program.
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.