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

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 e...

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Autores principales: Chato-Astrain, Jesús, Sánchez-Porras, David, García-García, Óscar Darío, Vairo, Claudia, Villar-Vidal, María, Villullas, Silvia, Sánchez-Montesinos, Indalecio, Campos, Fernando, Garzón, Ingrid, Alaminos, Miguel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8615600/
https://www.ncbi.nlm.nih.gov/pubmed/34829863
http://dx.doi.org/10.3390/biomedicines9111634
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author Chato-Astrain, Jesús
Sánchez-Porras, David
García-García, Óscar Darío
Vairo, Claudia
Villar-Vidal, María
Villullas, Silvia
Sánchez-Montesinos, Indalecio
Campos, Fernando
Garzón, Ingrid
Alaminos, Miguel
author_facet Chato-Astrain, Jesús
Sánchez-Porras, David
García-García, Óscar Darío
Vairo, Claudia
Villar-Vidal, María
Villullas, Silvia
Sánchez-Montesinos, Indalecio
Campos, Fernando
Garzón, Ingrid
Alaminos, Miguel
author_sort Chato-Astrain, Jesús
collection PubMed
description 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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spelling pubmed-86156002021-11-26 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, Indalecio Campos, Fernando Garzón, Ingrid Alaminos, Miguel Biomedicines Article 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. MDPI 2021-11-06 /pmc/articles/PMC8615600/ /pubmed/34829863 http://dx.doi.org/10.3390/biomedicines9111634 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Chato-Astrain, Jesús
Sánchez-Porras, David
García-García, Óscar Darío
Vairo, Claudia
Villar-Vidal, María
Villullas, Silvia
Sánchez-Montesinos, Indalecio
Campos, Fernando
Garzón, Ingrid
Alaminos, Miguel
Improvement of Cell Culture Methods for the Successful Generation of Human Keratinocyte Primary Cell Cultures Using EGF-Loaded Nanostructured Lipid Carriers
title Improvement of Cell Culture Methods for the Successful Generation of Human Keratinocyte Primary Cell Cultures Using EGF-Loaded Nanostructured Lipid Carriers
title_full Improvement of Cell Culture Methods for the Successful Generation of Human Keratinocyte Primary Cell Cultures Using EGF-Loaded Nanostructured Lipid Carriers
title_fullStr Improvement of Cell Culture Methods for the Successful Generation of Human Keratinocyte Primary Cell Cultures Using EGF-Loaded Nanostructured Lipid Carriers
title_full_unstemmed Improvement of Cell Culture Methods for the Successful Generation of Human Keratinocyte Primary Cell Cultures Using EGF-Loaded Nanostructured Lipid Carriers
title_short Improvement of Cell Culture Methods for the Successful Generation of Human Keratinocyte Primary Cell Cultures Using EGF-Loaded Nanostructured Lipid Carriers
title_sort improvement of cell culture methods for the successful generation of human keratinocyte primary cell cultures using egf-loaded nanostructured lipid carriers
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8615600/
https://www.ncbi.nlm.nih.gov/pubmed/34829863
http://dx.doi.org/10.3390/biomedicines9111634
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