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
Tissue Engineering Group, Department of Histology, Universidad de Granada, 18016 Granada, Spain.
Instituto de Investigación Biosanitaria ibs.GRANADA, 18012 Granada, Spain.
Biomedicines. 2021 Nov 6;9(11):1634. doi: 10.3390/biomedicines9111634.
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 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.
人皮肤角质形成细胞原代培养物可以从皮肤活检组织中建立，使用含有上皮生长因子（EGF）的培养基。尽管目前的方法很有效，但仍需要优化以加快程序并在更短的时间内获得这些培养物。在本研究中，我们评估了基于负载EGF的纳米结构脂质载体（NLC）的新型制剂的效果。首先，通过量化释放到培养基中的游离DNA，在永生化皮肤角质形成细胞、角膜上皮细胞以及两种上皮癌细胞系中验证了含有重组人EGF的NLC（NLC-rhEGF）的生物安全性。然后，我们用基础培养基（BM）以及补充了NLC-rhEGF、液体EGF（L-rhEGF）或单独的NLC（NLC-空白）的BM建立了人皮肤角质形成细胞的原代细胞培养物。结果表明，无论使用何种培养基，通过酶消化分离并在有或没有饲养层的情况下培养的细胞具有相似的生长速率。然而，与BM、L-rhEGF或NLC-空白相比，当使用NLC-rhEGF培养基时，外植体技术显示出更高的效率。基因表达分析表明，NLC-rhEGF能够增加基因表达，以及与细胞角蛋白、细胞间连接、角质形成细胞成熟和分化相关的其他基因的表达。总之，这些结果支持使用NLC-rhEGF来提高基于外植体的方法在高效生成用于组织工程的人角质形成细胞原代细胞培养物中的效率。
