Chato-Astrain, JesúsSánchez-Porras, DavidGarcía-García, Óscar DaríoVairo, ClaudiaVillar-Vidal, MaríaVillullas, SilviaSánchez-Montesinos, IndalecioCampos, FernandoGarzón, IngridAlaminos, Miguel2022-03-282022-03-282021-11-06Chato-Astrain J, Sánchez-Porras D, García-García Ó.D, Vairo C, Villar-Vidal M, Villullas S, et al. Improvement of Cell Culture Methods for the Successful Generation of Human Keratinocyte Primary Cell Cultures Using EGF-Loaded Nanostructured Lipid Carriers. Biomedicines 2021, 9 (11), 1634.http://hdl.handle.net/10668/3494Human 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.enAtribución 4.0 Internacionalhttp://creativecommons.org/licenses/by/4.0/EGFNanoparticlesNLCKeratinocytesTissue engineeringCell cultureEpidermal growth factorFactor de crecimiento epidérmicoNanopartículasQueratinocitosIngeniería de tejidosTécnicas de cultivo de célulaMedical Subject Headings::Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Primates::Haplorhini::Catarrhini::Hominidae::HumansMedical Subject Headings::Chemicals and Drugs::Hormones, Hormone Substitutes, and Hormone Antagonists::Hormones::Gastrointestinal Hormones::Epidermal Growth FactorMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Containment of BiohazardsMedical Subject Headings::Anatomy::Cells::Cells, Cultured::Feeder CellsMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Diagnosis::Diagnostic Techniques and Procedures::Clinical Laboratory Techniques::Cytological Techniques::Cell Culture Techniques::Primary Cell CultureMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Culture Techniques::Cell Engineering::Tissue EngineeringMedical Subject Headings::Chemicals and Drugs::LipidsMedical Subject Headings::Anatomy::Cells::Epithelial Cells::KeratinocytesMedical Subject Headings::Anatomy::Cells::Cells, Cultured::Cell LineMedical Subject Headings::Anatomy::Cells::Epithelial CellsMedical Subject Headings::Phenomena and Processes::Cell Physiological Phenomena::Cell Physiological Processes::Cell DifferentiationMedical Subject Headings::Phenomena and Processes::Genetic Phenomena::Genetic Processes::Gene ExpressionMedical Subject Headings::Anatomy::Cells::Cellular Structures::Cell Membrane::Cell Membrane Structures::Intercellular JunctionsMedical Subject Headings::Chemicals and Drugs::Chemical Actions and Uses::Specialty Uses of Chemicals::Laboratory Chemicals::Culture MediaMedical Subject Headings::Chemicals and Drugs::Macromolecular Substances::Polymers::Biopolymers::Intermediate Filament Proteins::KeratinsMedical Subject Headings::Phenomena and Processes::Digestive System and Oral Physiological Phenomena::Digestive System Physiological Phenomena::Digestive System Processes::DigestionMedical Subject Headings::Anatomy::Sense Organs::Eye::Anterior Eye Segment::Corneareview article34829863Acceso abierto10.3390/biomedicines91116342227-9059PMC8615600