Establishment And Biological Characterization Of Fibroblast Cell Lines Derived From Buffalo(Bubalus Bubalis)Fetus

Buffalo (Bubalus bubalis) is one of the most important domestic animals in Southern China, which was usually used as labor force for farming. But the potential economic value of Buffalo was gradually prominent with the deepening of relative research about it, especially using as the Bioreactor to produce target protein. Because there haven’t be established any Buffalo cell lines with the certain biological characterization yet. So it limits the deepened research of Buffalo in cell level and long-term preserve of the Buffalo resource. It also limits the research to abatain the thansgenic Buffalo with the nuclear thansfer method in which the cells constantly expressing target gene are transplantated into enucleated oocytes. So it is necessary to establish the Buffalo cell lines and concern their biological characterization.In this study,88Buffalo fetus from a local slaughterhouse in Nanning city were obtained and the tissues of ear margin、kidney and skin were cultured. The primay cells were separated and purified to obtain the fibroblast. And thenthese fibroblast cell lines were used for the analysis of growth kinetic assay, survival rate before freezing/after being thawn, contamination of microbiology, karyotype, Isoenzyme, fluorescent protein expression rate and cell cycle. Finally, two skin fibroblast cell lines (BFSF101107、 BFSF101007), one ear margin fibroblast cell line (BFEF), one kidney fibroblast cell line and one kidney fibroblast cell line with a flat form were established from all the cells obtained. The growth curve of those cell lines are "S" shape, which were drawn according to the result of cell growth kinetic assay. These cell lines had a higher survival rate before cryopreservation and after recovery. The results of micro-organism contamination detection were negative. Next, these cell lines were detected if there was any cross contamination by the analysis of chromosome karyotype and isozyme. Most of the cells detected had48chromosomes each (cells used in this assay are in the20th passage); the pattern of isozyme analysis indicated that there was no obvious evidence about cross contamination between those cell lines. After being transfected GFP and RFP plasmid respectively under the same condition, BFSF101107had a much higher expression rate and BFKF-Flat seems to express more fluorescent protein than other cell lines. And the cell cycle analysis was adopted to confirm chromosome homogeneity and chromosome mutation probability of cell lines in20th generation. The result indicated that BFSF101107is better than the others. So these cell lines with different biological characterization can be choosed for dreepened research on cell level with different acquirement of the characterization.Successfully, we have passed the all the cell lines above for30times with antibiotic-free culture system, and established two Buffalo fetus skin fibroblast cell lines(BFSF101107、BFSF101007)、one Buffalo fetus ear margin fibroblast cell lines (BFEF)、one Buffalo fetus kidney fibroblast cell line、one flat-like Buffalo fetus kidney fibroblast cell line. Considering all the results of the cell assay, the fibroblast cell derived form skin is more optimal compared with the other tissue-source fibroblast cells. It provides a reference for cell lines establishment of large domestic animal.