The Establishment And Biological Characteristics Of Chicken Embryo Cecal Epithelial Cell Line

In order to explore the invasion and damage mechanism of Eimeria tenella on host cells and provide an in vitro cell model for the study of anticoccidial drugs,this study isolated and established a chicken embryo cecal epithelial cell line from SPF chicken embryo cecum.The chicken embryo cecal epithelial cell line was cultured by determining the selection of different concentrations of growth factors in the cell culture medium.The study on identification and biological characteristics of chicken embryo cecal epithelial cell line through using of optical and transmission electron microscopy,cell growth curve,population doubling time,cell viability before and after freezing and thawing,expression of epithelial cell marker protein,karyotype analysis,isoenzymes detection,mycoplasma detection,Eimeria tenella infection rate,etc.The results showed that:(1)The best concentration of growth factor for chicken embryo cecal epithelial cells culture medium was insulin 10 μg/m L,hydrocortisone 1.5 μg/m L,transferrin 5 μg/m L and fibronectin 1 μg/m L.It was successfully established a chicken embryo cecal epithelial cell in vitro culture system by using the above-mentioned medium to culture the primary and passage cells.(2)The morphology of chicken embryo cecal epithelial cells was observed by inverted microscope.It was found that the crypt cells attached to the culture plate and began to stretch out the cecal epithelial cells on 1 day after inoculation of the primary chicken embryo cecal epithelial cells.The cells had clear boundaries and tight connections,showing a typical "paving stone-like" shape on 2-3 days.The cells grew fast and most of the cells grew in an island shape on 4-6 days.The cells covered the culture plate on 7-8days.Passage continuously to the 15 th generation,the island-like aggregated cells gradually decreased.The hallmark structures of chicken embryo cecal epithelial cells,such as microvilli,tight junction structure and desmosomes and the morphology of mitochondria and endoplasmic reticulum were good,which can be observed under a transmission electron microscope.(3)The cell viability was measured by MTT.The growth curves of primary,5th,10 th and 15 th generation chicken embryo cecal epithelial cells were drawn.They all showed a typical "S" shape.The cell population doubling time was 20.62 h,which was in line with the cells general growth pattern.Serum:DMSO 9:1 was used as the cryopreservation solution combined with the gradient cooling method to freeze the cells in liquid nitrogen.The cells were quickly thawed in a 37℃ water bath during resuscitation.The results showed that the cell viability was not significantly different before and after cryopreservation(P >0.05).It shows that the chicken embryo cecal epithelial cells have good biological characteristics.(4)The primary and 15 th generation chicken embryo cecal epithelial cells were stained with cytokeratin-18 and E-cadherin,and the identification was positive by using immunofluorescence staining.The expression of epithelial cytokeratin-18 and E-cadherin genes were detected in the 5th,10 th and 15 th generation chicken embryo cecal epithelial cells by using RT-PCR.It shows that the cultured cells are epithelial cells.(5)The results of karyotype analysis showed that both the primary and 15 th generation chicken embryo cecal epithelial cells were diploid karyotypes,which were genetically stable.Isoenzymes detection of primary and 15 th generation chicken embryo cecal epithelial cells and chicken macrophages had the same malate dehydrogenase and nucleotide phosphate dehydrogenase zymograms.Furthermore,there were no other miscellaneous bands,indicating that the cells are the same species as chicken macrophages,and there is no cross-contamination of other cells.The results of mycoplasma detection was negative indicating that the cultured chicken embryo cecal epithelial cells are not contaminated by mycoplasma.The infection rate of Eimeria tenella in the 15 th passage of chicken embryo cecal epithelial cells was not significantly different from that of primary cells(P > 0.05).It shows that the chicken embryo cecal epithelial cell line can be used as an in vitro culture model for coccidia-related research.