Establishment And Validition Of Three Human Embryonic Lung Fibroblast Cell Lines

Vaccines are not only national strategic needs,but also an important guarantee for life and health.Cell substance determines the safety and efficacy of viral vaccines,so it is the key to vaccine development and production.Human diploid cells are the ideal matrix for viral vaccine production.At present,the resources of human diploid cell lines approved for use in China have gradually decreased,the cell generation is relatively high,and the cell generation that can be used for vaccine production is limited,which makes it difficult to meet the actual demand of large-scale production.Therefore,innovative research,which establish human diploid cell lines that meet the production requirements and prolonging the life span of human embryonic lung cells,for the high-quality development of viral vaccine industry.After ethical review,this study collected healthy human embryonic lung tissue from induced abortions,obtained three primary human embryonic lung fibroblast cell lines through isolation and cultivation,and stored them in a cell bank.This study determined the culture conditions of human embryonic lung cells,and completed the overall quality evaluation of the cell bank,including the growth characteristics,genetic characteristics,internal and external pollution factors,tumorigenicity and virus sensitivity.Meanwhile,the study used lentiviral transfection to introduce the SV40-LT gene into human embryonic lung cells,establishing a stable cell line overexpressing SV40-LT（MU-SVLs）to extend the lifespan of human embryonic lung cells and establish a human embryonic lung immortalized cell lines.The study also investigated the stability of SV40LT gene expression,growth characteristics,genetic characteristics,tumorigenicity,and support for virus proliferation in the MU-SVLs cell lines.1.Three human embryonic lung fibroblast cell lines（MU-Ls）were isolated from aborted embryonic lung tissues with gestational age of 15W,11W+5 and 11W+1 by pancreatin digestion method,and were named as MU-L1,MU-L2 and MU-L3 cells successively.This study determined that the culture condition of human embryonic lung fibroblast cells,that MEM medium supplemented with 10%（v/v）newborn bovine serum and the subculture ratio of 1:4.MU-L1,MU-L2 and MU-L3 cells were continuously cultured under this culture condition,and attained to the passages of 67,77 and 75,respectively.In the process of continuous passage,the cells were frozen in liquid nitrogen every 10 passages,and the viability of the cells after resuscitation was kept above 85%,and the highest was above 99%.2.The growth curve was drawn by the digestive counting method to compare the growth characteristics of the three strains of human embryonic lung fibroblast cells in different lifetimes.It was found that,the proliferation ability was weakened with the increase of cell generations.The positive rate ofβ-galactosidase staining increased with higher cell generation.All the three cells were normal diploid karyotype,and the karyotype 2n=46 of passages 11 and 31,was more than 90%.The vimentin expression of the three cells was positive and showed fibroblast-like expression.The test results showed that no bacteria,fungi,mycoplasma and internal and external viral factors were found in the three cells.The three cell lines were sensitive to EMCV,and the TCID50/0.1ml were 10^-6.53±0.41,10^-6.98±0.37and 10^-6.28±0.3,respectively,after 96h inoculation,which were higher than the standard control cell MRC-5(10^-5.78±0.2).3.By lentivirus transfection technique,SV40-LT gene was introduced into human embryonic lung fibroblast cells MU-L1（P24）,MU-L2（P9）and MU-L3（P9）,and three human embryonic lung fibroblast cell lines overexpressing SV40-LT gene were established,which were successively named as MU-SVL1,MU-SVL2 and MU-SVL3.On the basis of the original generation,it has been cultured to Passages 90,91and 89,respectively.RT-PCR and Western Blot showed that SV40-LT gene was highly expressed at Passages 85.MU-SVLs cells had a diploid karyotype（2n=46）,and the incidence of polyploid（2n>53）increased with the increase of cell generation.The results of tumor carrying experiment in nude mice showed that none of the MU-SVLs cells were neoplastic.This study successfully isolated and cultured three human embryonic lung fibroblast cell lines with good biological characteristics,normal karyotype,and non-tumorigenicity,which are sensitive to EMCV and have value for further research and production.By using lentiviral infection technology,three human embryonic lung fibroblast cell lines stably overexpressing SV40-LT were constructed,and their lifetimes were significantly prolonged.It is possible to build immortalized human embryonic lung.