Abstract
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.
Highlights
In the present study we evaluated the potential of human recombinant EGFloaded nanostructured lipid carriers (NLC) to improve methods currently available to generate primary cultures of human skin keratinocytes, with the ultimate goal of shortening the time required to produce an efficient skin substitute by tissue engineering
Twenty-four hours later, the culture medium was replaced with basal culture media (BM), liquid recombinant human EGF (L-rhEGF), NLC-rhEGF, or NLC-blank, and cells were incubated for 10 days under standard culture conditions
To determine the effect of each type of culture medium on gene expression, human keratinocytes cultured with BM, L-rhEGF or NLC-rhEGF were analyzed by RT-PCR
Summary
One of the main challenges in the treatment of patients with severe burns is to achieve rapid, effective closure of the skin injuries to prevent fluid loss and nosocomial infections, the two major complications in these patients [7,8,9] For this reason, further research focused on shortening the time required to fabricate bioartificial skin is needed. In the present study we evaluated the potential of human recombinant EGF (rhEGF)loaded NLC to improve methods currently available to generate primary cultures of human skin keratinocytes, with the ultimate goal of shortening the time required to produce an efficient skin substitute by tissue engineering
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
