Effects Of Different Culture Medium And Feeder Cells On The Culture Of Echinococcus Multilocularis In Vitro

BACKGROUND Cystic culture of Echinococcus multilocularis protoscolex in vitro is the experimental basis for extracting and studying germinal layer stem cells.D-MEM and RPMI-1640 are the most commonly commercial media used for Culture of Echinococcus multilocularis in vitro,and HeLa cells and rat hepatoma cells(ATCC No.CRL-1600)are also commonly used feeder cells.The effects of the combination of these two media and feeder cells on the growth of Echinococcus multilocularis in vitro have not been studied directly.OBJECTIVE A culture model of Echinococcus multilocularis in vitro was established to observe the growth of Echinococcus multilocularis in different feeder cells(HeLa cells and rat hepatoma cells(ATCC no.CRL-1600)and different media(D-MEM(High and Low glucose type)and RPMI-1640)containing 10% fetal bovine serum,providing a new idea for obtaining alveolar cysts from Echinococcus multilocularis in vitro.METHODS HeLa cells and rat hepatoma cells(ATCC No.CRL-1600)were pre-cultured.The proscolex of Echinococcus alveolaris was extracted from the mice fed for more than 6 months and co-cultured with different feeder cells and culture media.The experiment was divided into four groups: group I: HeLa cells and RPMI-1640 medium;group II: HeLa cells and D-MEM(High glucose type)medium;group III: hepatocellular carcinoma cells and RPMI-1640 medium;group IV: hepatocellular carcinoma cells and DMEM(High glucose type)medium;V: HeLa cells and D-MEM(Low glucose type)medium;VI: hepatocellular carcinoma cells and D-MEM(Low glucose type)medium observe the survival,growth and development of the protoscolex,calculate the rate of protoscolex vesicle formation every 7 days,and measure the average diameter of vesicle with microscope micrometer.Results protoscolex could survive for a long time in all four combinations.The vesicle diameter ranged from 1 mm to 6 mm.On the 56 th day of culture,the cyst formation rate and average vesicle diameter of protocephalus were the best in the group of rat hepatocellular carcinoma cells and D-MEM(high sugar type)medium(P < 0.05),the cyst formation rate was 72.08(+1.79)%,and the average vesicle diameter was 3.379(+0.199)mm.Quantitative protein detection results showed that the contents of vesicle fluid protein in 6 groups were lower than those of vesicles in vivo,and the contents of histone IV were higher than those of other 5 groups(P < 0.05).Conclusion:1.Under the same conditions,the vesicle size and quantity of D-MEM(high sugar type)medium were better than those of the other two media,Glucose content in culture medium correlated with vesicle growth rate.2.Rat hepatoma cells(ATCC No.CRL-1600)as feeder cells are superior to HeLa cells.3.D-MEM(High glucose type)combined with rat hepatoma cells(ATCC No.CRL-1600)has the highest cyst formation rate and vesicle size among the four groups,and can be used as the best choice for the culture of alveolar echinococcus vesicles in vitro.