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...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	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
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	EGF nanoparticles NLC keratinocytes tissue engineering cell culture Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/6a79f643bb274096812a1c35eeb1562a
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:6a79f643bb274096812a1c35eeb1562a
record_format	dspace
spelling	oai:doaj.org-article:6a79f643bb274096812a1c35eeb1562a2021-11-25T16:49:52ZImprovement of Cell Culture Methods for the Successful Generation of Human Keratinocyte Primary Cell Cultures Using EGF-Loaded Nanostructured Lipid Carriers10.3390/biomedicines91116342227-9059https://doaj.org/article/6a79f643bb274096812a1c35eeb1562a2021-11-01T00:00:00Zhttps://www.mdpi.com/2227-9059/9/11/1634https://doaj.org/toc/2227-9059Human 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 <i>EGFR</i> 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.Jesús Chato-AstrainDavid Sánchez-PorrasÓscar Darío García-GarcíaClaudia VairoMaría Villar-VidalSilvia VillullasIndalecio Sánchez-MontesinosFernando CamposIngrid GarzónMiguel AlaminosMDPI AGarticleEGFnanoparticlesNLCkeratinocytestissue engineeringcell cultureBiology (General)QH301-705.5ENBiomedicines, Vol 9, Iss 1634, p 1634 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	EGF nanoparticles NLC keratinocytes tissue engineering cell culture Biology (General) QH301-705.5
spellingShingle	EGF nanoparticles NLC keratinocytes tissue engineering cell culture Biology (General) QH301-705.5 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 Improvement of Cell Culture Methods for the Successful Generation of Human Keratinocyte Primary Cell Cultures Using EGF-Loaded Nanostructured Lipid Carriers
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 <i>EGFR</i> 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.
format	article
author	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
author_facet	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
author_sort	Jesús Chato-Astrain
title	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_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_sort	improvement of cell culture methods for the successful generation of human keratinocyte primary cell cultures using egf-loaded nanostructured lipid carriers
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/6a79f643bb274096812a1c35eeb1562a
work_keys_str_mv	AT jesuschatoastrain improvementofcellculturemethodsforthesuccessfulgenerationofhumankeratinocyteprimarycellculturesusingegfloadednanostructuredlipidcarriers AT davidsanchezporras improvementofcellculturemethodsforthesuccessfulgenerationofhumankeratinocyteprimarycellculturesusingegfloadednanostructuredlipidcarriers AT oscardariogarciagarcia improvementofcellculturemethodsforthesuccessfulgenerationofhumankeratinocyteprimarycellculturesusingegfloadednanostructuredlipidcarriers AT claudiavairo improvementofcellculturemethodsforthesuccessfulgenerationofhumankeratinocyteprimarycellculturesusingegfloadednanostructuredlipidcarriers AT mariavillarvidal improvementofcellculturemethodsforthesuccessfulgenerationofhumankeratinocyteprimarycellculturesusingegfloadednanostructuredlipidcarriers AT silviavillullas improvementofcellculturemethodsforthesuccessfulgenerationofhumankeratinocyteprimarycellculturesusingegfloadednanostructuredlipidcarriers AT indaleciosanchezmontesinos improvementofcellculturemethodsforthesuccessfulgenerationofhumankeratinocyteprimarycellculturesusingegfloadednanostructuredlipidcarriers AT fernandocampos improvementofcellculturemethodsforthesuccessfulgenerationofhumankeratinocyteprimarycellculturesusingegfloadednanostructuredlipidcarriers AT ingridgarzon improvementofcellculturemethodsforthesuccessfulgenerationofhumankeratinocyteprimarycellculturesusingegfloadednanostructuredlipidcarriers AT miguelalaminos improvementofcellculturemethodsforthesuccessfulgenerationofhumankeratinocyteprimarycellculturesusingegfloadednanostructuredlipidcarriers
_version_	1718412901918703616

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

Ejemplares similares