Deficiency in SI protein results in osmotic diarrhea due to an inability to hydrolyze intestinal disaccharides into component monosaccharides. Therefore, we conclude that the newly established cell line is comprised of small intestine-derived epithelial cells, which in turn suggests that this cell line can be used in future research on disease inducing porcine diarrhea. Typically, differentiated cells were taken for representing the intestinal villus tip cells, while the undifferentiated cells were used to mimic the basilar crypt cells of the intestines. Thus, the prototypical characteristics of differentiated porcine intestinal epithelial cells are the presence of tight junctions and distinct microvilli on their apical surfaces. Here, we demonstrated that porcine intestinal epithelial cells expressed markers typical of differentiated enterocytes, specifically, ZYM-SIEC02 cells were positive for E-cadherin, ZO-1, Occludin, villin, and sucrose isomaltase, which is the most reliable indicator of intestinal cell differentiation in vitro, indicating the presence of differentiated villus cells in the culture. IPEC-J2 cells were cultured for 1 days or 21 days, representing undifferentiated proliferating and highly differentiated IPEC-J2 cells, respectively. However, in our experiment, all the ZYM-SIEC02 cells we used were cultured to have a confluent monolayer within 3 or 6 days according to the density of cell cultures. In this regard, the characteristics of differentiated ZYM-SIEC02 cell were evidently due to the time that isolated and purified the primary porcine intestinal epithelial cells was long enough for the differentiation of pSIECs, which prepared for the lipofection. Thus, ZYM-SIEC02 cells remain the characteristic of differentiated pSIECs, and markers typical of differentiated Cycloheximide enterocytes were detected. Moreover, we used tissue culture method to isolate epithelial cells from small intestine, it was unavoidable that the growth of fibroblasts over cultured primary intestinal epithelial cells, however, on the other side of the coin, the intestinal epithelial cell differentiation need the heterologous cell-cell contacts for denovo synthesis to trigger cell polarity and differentiation. The addition of EGF may be another important inducement for differentiation as it plays a pivotal role in the regulation of intestinal epithelial proliferation and differentiation. Over time in culture, cells maintained their epithelial morphology as well as expression of markers. These characteristics were not affected by cryogenic freezing and retrieval. An early study by Hayflick et al demonstrated that normal human somatic cells have a finite replicative potential in vitro, and will stop dividing after a finite number of population doublings and enter senescense.