Creation Of An In Vitro Model Of Differentiated Swine Trachea Epithelial Cell Culture

Swine tracheas were lined by different types of pseudostratified epithelial cells.They constist of ciliated cells,goblet cells,and basal cells.In order to study the complex mechanism and different functions of epithelial cells,researchers have developed model of culture primary human epithelial cells,used a method of air-liquid interface to culture primary trachea epithelial cells('I EC).The morphology of this in vitro airway epithelial cells similar to in vivo growth and can be used to study the virus and other respiratory pathogens of swine trachea infection model.However,due to the cell in vivo 3 dimention(3D)environment,they're not differentiated into functional morphology.Therefore,in vitro epithelial cells differentiated into functional ones is still a difficulty in the model.In order to establish the culture model of primary swine primary trachea epithelial cells(STEC)cultured in air-liquid interface,we screened to determine the selection criteria of donor pig,digestion of primary cell separation time,culture medium formula.We recommend to use of 2-3 months old pigs with no porcine circovirus type 2,porcine reproductive and respiratory comprehensive syndrome virus or Mycoplasma hyopneumoniae and other respiratory tract pathogens infected.For 2-3 month old pigs,the optimal digestion time of tracheal epithelial cells will be 24-30 hours.For medium,a modified bronchial epithelial growth medium(BEGM)with a final concertration of epidermal growth factor is lOng/ml,the retinoic acid is 10'7M is the best.After screening of differert kinds of mediums,BEGM,modified BEGM/5%FCS,DMEIM/F12/2%USG and DMEM/F12/5%FCS,we found that the modified BEGM/5%FCS was suitable for cell's attachment and proliferation.By using this medium to culture swine primary airway epithelial cells for 24 hours,the rate of the attached cells were high,the proliferations of cells were also improved.In general,STEC can be rapidly covered with culture cell membrane surface in 2-4 days and create an air liquid interface;When cells grown 6 days in modified BEGM/5%FCS and scanned with electron microscope(SEM),most of the STECs were covered microvilli,a few of unmature cillia can be seen.The cells cultured in an air-liquid interface for 7 days,they showed circular stereo shapes,the transepithelial electric resistance(TEER)reach 2800/cm2;14 days later,from the SEM,we observed of more mature ciliated cells;but when the cells cultured for 21 days,most of the cells begin to flote.A few of remaining cells has differentiated into mature cilia.This study successfully established the model of air-liquid interface method to culture primary porcine airway epithelial cells.The next step will focus on the duration and long term to maintain the STECs and the establishment of standard operating procedure of the cell culture model.