Isolation of human primary gastric mucosa epithelial cells

1. A piece of gastric mucosa (∼3 cm²) was obtained from the normal appearing mucosa of the stomach at surgery.

2. The patients were proven to be no H. pylori-negative by a combination of serology, histology and culture.

3. The patients gave informed consent, and the Human Research Committee approved the study.

4. The surface mucosal layer was removed with a razor blade, immediately minced, and then incubated in Ham's F-12 culture medium containing collagenase type I (0.2 mg/ml; Invitrogen) for 10 min.

5. Cells from the final incubation were washed and cultured in Ham's F-12 medium supplemented with 10% FBS and streptomycin (300 μg/ml) at 37°C in a humidified 5% CO^₂ atmosphere.

6. Epithelial cells were cultured in a 24-well collagen-coated dish at a final concentration of 106 cells/ml for 24 h before use.

7. Cultured cells formed subconfluent monolayers within 24 h of the inoculation.

8. The cultured cells in the monolayers had periodic acid-Schiff-positive material in the cytoplasm, confirming that the population consisted of mucus producing epithelial cells with at most a minimal contamination by other cells.

9. Each experiment used gastric cells from a different patient with individual experiments being performed by using gastric cells from a single patient.

10. The results were pooled from three different experiments.

Reference

1. Tanahashi, T., Kita, M., Kodama, T., Yamaoka, Y., Sawai, N., Ohno, T., Mitsufuji, S., Wei, Y. P., Kashima, K., and Imanishi, J. (2000). Cytokine expression and production by purified Helicobacter pylori urease in human gastric epithelial cells. Infect. Immun. 68, 664-671.
2. Smoot D. T., Resau J. H., Naab T., Desbordes B. C., Gilliam T., Bull H. K., Curry S. B., Nidiry J., Sewchand J., Mills R. K. (1993) Adherence of Helicobacter pylori to cultured human gastric epithelial cells. Infect. Immun. 61:350–355.
3. Takahashi M., Ogura K., Maeda S., Mori K., Mafune K., Mikami Y., Terano A., Omata M. (1997) Promoters of epithelialization induce expression of vascular endothelial growth factor in human gastric epithelial cells in primary culture. FEBS Lett. 418:115–118.