Application Of Nanoself-Assembling Peptide RADA16-I In Three-Dimensional Cell Culture Model Of Adenovirus

Objective: To construct three-dimensional(3D)culture model of adenovirus using nanoself-assembling peptide RADA16-I as 3D cell culture scaffold in vitro,and combining with virology experimental technology,providing a novel research method for virus isolation and culture,pathogenesis research,and antiviral drug screening.Methods:(1)The structural characteristics of nanoself-assembling peptide RADA16-I were characterized by SEM and TEM.(2)The 3D culture model of 293 T cells was constructed by nanoself-assembling peptide RADA16-I as scaffold in vitro,then the activity of 293 T cells was analyzed in the 3D cell culture system.The main observation indicators were as follows:(1)The morphology of 293 T cells was observed using inverted phase contrast microscope.(2)Morphology and activity of3D-cultured 293 T cells were observed by Ca-AM,DAPI and PI staining.(3)The proliferative activity of 293 T cells in 3D cell culture system was detected by MTS assay.(3)Adenovirus with enhanced green fluorescent protein(EGFP)label target was inoculated in the 3D cell culture system,then the cultivation of 293 T cells and adenovirus in this model were analyzed.The main observation indicators were as follows:(1)Morphology of infected-293 T cells in the 3D system was observed by fluorescence inverted microscope.(2)The proliferative activity of 3D-cultured 293 T cells was detected by MTS assay at different time points after infection with adenovirus.(3)Adenovirus proliferation in 3D cell culture system was analyzed through the number of green fluorescent cell masses.(4)Adenovirus virions in 3D cell culture system were observed through TEM assay.(5)Viral propagation in 3D cultures and infectivity of virus particles produced from the 3D system were measured using q PCR assay.(6)TNF-? and IL-8 in the media of 3D cultures were examined by ELISA at the different time points after infection with adenovirus.Results:(1)RADA16-I material self-assembled into highly cross-linked nanofibers which interwine each other to form a 3D network.(2)The 3D culture system using RADA16-I as scaffold was suitable for the growth of 293 T cells:(1)Individual 293 T cells cultured in RADA16-I hydrogel gradually formed multicellular spheroids.(2)With the extending of cultivate time,the spheroids formed by 3D-cultured 293 T cells gradually enlarged and kept well viability.(3)Cell proliferation of 293 T cells in3 D system kept multiplying for a long time and the proliferation activity peaked on11-15 days after cultivation.(3)The growth of adenovirus and 293 T cells in RADA16-I 3D system:(1)Vesicle formation of 3D multicellular spheroids was visible at 5-6 dpi and cytolysis was observed at 9-12 dpi,at which point individual cells were visible.(2)Green fluorescent cell masses in 3D culture were observed at 60-72 hpi,but the fluorescence intensity was weak.Thereafter the number of fluorescent cell masses and fluorescence intensity increased gradually from the edge of the gel,peaking on 8dpi,then decreased gradually and peaked on 16 dpi and 22 dpi,respectively.By contrast,the green fluorescence of cells in 2D culture was visible at 24 hpi and the fluorescence intensity was strongest at 48-72 hpi.In addition,apparent CPEs could be observed and the cells gradually became necrotic and detached after 4-6 dpi.(3)A small number of virions whose diameter at 60-90 nm were significantly observed in the cytoplasm of 3D-cultured 293 T cells at 9 dpi and 15 dpi.(4)Multiple spikes appeared in adenovirus DNA levels,whose proliferation characteristics was consistent with the result of green fluorescence cell masses counting.Viral DNA levels increased significantly at the 8 dpi and then then decreased gradually and peaked on 16 dpi and22 dpi.Meantime,the virons produced from the 3D system remained infectious.(5)Secretion levels of TNF-? and IL-8 appeared as multiple peaks in 3D cultures after infection with adenovirus,but the expression levels of TNF-? and IL-8,as well as the secretion trends,were different in the cell cultures.Conclusion: RADA16-I can be used to construct a 3D culture model of adenovirus in vitro,which is expected to provide a novel research method for adenovirus isolation and culture,pathogenic mechanism research and the antiviral drugs screening.