Establishment Of Intestinal Epithelial Cell Line From Neonatal Goat

Animal cell and tissue culture has been widely used in cell biology and molecular biology, especially on the study of mechanism of cell differentiation. Small intestine is the main place for digestion and absorption of nutrient. Intestinal epithelial cells (IEC) are the primary functional cells in intestinal tract. Besides participating in digestion, absorption, immunization barrier and stress reaction, IECs are closely related to secretory function internal and external intestine. As a result, isolation and culture of intestinal epithelial cells is one of major means to study on intestine function, pathology and cell differentiation,nutrient absorption and regulatory mechanism.Both methods of explant culture and enzymatic digestion were used in this study to separate epithelial cells from intestine of neonatal goat. The result showed that the viable epithelial cells which have high capacity of proliferation can be observed by means of explant culture method. And the epithelial cell line can be obtained through scraping, which could passage up to 12 generation steadily. Isolation of epithelia and preservation of its integrity was well achieved by using collagenase (300U/ml)/dispase (0.1mg/ml) digestion technique, the epithelial cell attached as intact units (villi and crypt units). The epithelium in these organoid units remained as polarized intact layers, and attached readily to collagen coated dishes within 24h, forming large coalescing colonies of epithelium within 48h. But, it was difficult for the epithelial cells to attach and the multiplication capacity decreased after passage. The cells obtained by 0.25% trypsin and collagenase digestion were mostly fibroblasts which were difficult to attach. The thermolysin digestion method could generated viable epithelium, but it was hard to attach and proliferate after passage.MTT colorimetric assay was developed to allow a reproducible and quantitative measurement to determine the effects of fetal bovine serum (FBS), glutamine (Gln), epidermal growth factor (EGF) and insulin on the growth of IEC from neonate goat The results indicated that the optimum condition for IEC culture were: 10% FBS,2 mmol/ml Gln,10 ng/ml EGF and 5μg/ml insulin. In this study, two kinds of frozen stock solution were used to stock IEC, result showed that supplement of dextran could protect IEC during the process of stock, and increase the livability of stock cells.