Isolation of human colonic epithelial cells (EC)

                      Isolation of human colonic epithelial cells (EC)       
1.Human colonic epithelial cells (EC) were isolated from freshly resected colonic surgical specimens obtained.

2.All diagnoses were confirmed by clinical, radiological, endoscopic, and histological findings.

3.Solid tissues were minced with scissors into small (2-mm3) fragments and incubated for 15 min at room temperature in 100 mM phosphate buffer (pH 7.0) with 6.5 mM DTT to remove mucus contamination.

4.Mucosal strips were treated at room temperature for 30 min in 1.5 mg/ml DTT and then gently stirred in a 1-mM EDTA solution for three 60-min periods.

5.After gentle removal of the DTT solution, tissue fragments were rinsed once with Hank's balanced salt solution (HBSS), resuspended in serum-free primary cell culture medium (2 mmol/liter l-glutamine, 120 μg/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml ceftazidime, 0.25 μg/ml amphotericin-B, 20 mmol/liter Hepes) with 200 units/ml Collagenase type III and 100 units/ml DNase I, and incubated for 2 h at 37℃ to obtain enzymatic disaggregation.

6.The supernatants were collected and centrifuged, and the resulting pellets were suspended in primary cell culture medium.

7.Cell suspensions were digested with 3 mg/ml dispase and 0.5 mg/ml DNase I from Primary cell isolation kit for 30 min at 37℃.

8.Cells were then resuspended by pipetting and serially filtered by using sterile gauze and 70-μm and 40-μm nylon meshes.

9.Contaminating red blood cells were removed by osmotic lysis.

10.ECs were purified over a 50% Percoll gradient, spun at 1,500 rpm for 20 min.

11.ECs equilibrated at the interface were collected and washed.

12.The purity and viability of ECs were consistently >97%, with <3% contaminating mononuclear cells.