The aim was to investigate a possibility of using the cryopreserved human culture of fibroblasts (CrHFC) with gold nanoparticles (AuNPs) to treat experimental burns in rats.The third-degree burns were modeled in white male rats. All the animals with burns were divided into three experimental groups: control group with no wound treatment; group 1 was composed of animals with CrHFC application; and group 2 consisted of those with CrHFC and AuNPs (6 μg/ml) application to a burn surface the next day after the injury. The CrHFC was applied to the methylcellulose gel in a dose of 5 × 104 of viable cells per 1 cm2 of the burn. The animals were removed from the experiment on day 21 after the treatment.The CrHFC use alone and with AuNPs to the surface of burns stimulated the wound healing compared with the control. The effect of using CrHFC was less pronounced compared to the CrHFC application with AuNPs. It was reflected in a slower recovery of burns and moderate lymphocytic infiltration of granulation tissue. Immunofluorescent analysis emphasized that the use of CrHFC with AuNPs accelerated the skin synthetic processes and was helpful in recovering type I and III collagen content on day 21 after therapy.The results were likely related primarily to the unique structure and antimicrobial properties of AuNPs. Our experimental study of the effect of CrHFC with AuNPs application on regenerative processes in burns gives some pre-conditions to the following advanced bio- and nanotechnology developments.

Background: CRISPR/Cas9 system is becoming the dominant genome editing tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific. Methods: Three gRNAs were chosen to target chicken 3’-untranslated region of GAPDH gene. Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along with CRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene ( neoR) under cytomegalovirus promoter. Correct integration of the cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS). Results: We have established a CRISPR/Cas9 system to target an endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of the chicken GAPDH promoter. Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.

Cell culture models are excellent tools for potential toxicity of nanoparticles and fundamental investigations in cancer research. Thus, information about AuNP potential toxicity and effects on human health is necessary for the use of nanomaterials in clinical settings. The aim of our research is to examine the effects of AuNPs on the epithelial origin cell lines: continuous and oncogenic. Embryonic porcine kidney epithelial inoculated (SPEV) cell line and colorectal carcinoma cell line (HT29) were used. In the test cultures, the cell proliferation, necrosis/apoptosis, and multicellular spheroids generation were evaluated. We demonstrated that AuNP concentrations of 6–12 μg/ml reduced the proliferation of SPEV and HT29 cells and increased the cell number at early and late stages of apoptosis and necrosis. It was shown that small concentrations of AuNPs (1–3 μg/ml) stimulate multicellular spheroid formation by HT29 and SPEV cells. However, higher AuNP concentrations (6–12 μg/ml) had both cytotoxic and anti-cohesive effects on cell in suspension. The large sensitiveness to the action of AuNPs was shown by the line of HT29 (6 μg/ml) as compared to the SPEV cells (12 μg/ml). This experimental study of the effect of AuNPs on SPEV and HT29 cell lines will justify their further application in AuNP-mediated anticancer treatment.

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