Research on two culture methods for bronchia epithelia.

Objective To explore two technologies of separation and culture for pig trachea-bronchia epithelia: traditional culture and air-liquid interface culture,look for appropriate freezing and anabiosis conditions of the epithelia and combine advantages of the two culture technologies.Method Pig trachea-bronchia epithelia was separated,purified and kept passage.The livability of descended epithelia was calculated by the assay of freezing and anabiosis to look for best freezing and anabiosis conditions.After anabiosis the epithelia were cultured by air-liquid method and its growth characteristics and the appearance of the cells′ cilia were observed.The growth curve was also made.Finally the test of keratins of pig trachea-bronchia epithelia was done to identify the epithelia.Result Through four-step purification highly pure epithelia were gotten.The average epithelia livability was 89% after anabiosis,which was freezed with a system including 50% of Fetal Serum,40% of DMEM/F12 and 10% of DMSO.The epithelia passage number was eight with the traditional culture and after the second generation the epithelia cilia became invisible.The cilia of some epithelia cultured by air-liquid method came into being after continuously descended twice and the epithelia's suvival time was also prolonged.The result of the test of keratins indicated the cultured cells were epithelia.Conclusion We have successfully established two culture technologies and found the appropriate freezing and anabiosis technology for pig trachea-bronchia epithelia.The epithelia by traditional culture,after freezing and anabiosis,can successfully adapt to air-liquid culture and recover their natural morphological characteristics.The advantages of the two culture technologies have been successfully combined,which provide a rich cell source for researches of pig trachea-bronchia epithelia.