Establishment of a three-dimensional culture system of gastric stem cells supporting mucous cell differentiation using microfibrous polycaprolactone scaffolds

Objectives: To generate various polycaprolactone (PCL) scaffolds and test their suitability for growth and differentiation of immortalized mouse gastric stem (mGS) cells. Materials and methods: Non-porous, microporous and three-dimensional electrospun microfibrous PCL scaffolds were prepared and characterized for culture of mGS cells. First, growth of mGS cells was compared on these different scaffolds after 3 days culture, using viability assay and microscopy. Secondly, growth pattern of the cells on microfibrous scaffolds was studied after 3, 6, 9 and 12 days culture using DNA PicoGreen assay and scanning electron microscopy. Thirdly, differentiation of the cells grown on microfibrous scaffolds for 3 and 9 days was analysed using lectin/immunohistochemistry. Results: The mGS cells grew preferentially on microfibrous scaffolds. From 3 to 6 days, there was increase in cell number, followed by reduction by days 9 and 12. To test whether the reduction in cell number was associated with cell differentiation, cryosections of cell-containing scaffolds cultured for 3 and 9 days were probed with gastric epithelial cell differentiation markers. On day 3, none of the markers examined bound to the cells. However by day 9, approximately, 50% of them bound to N-acetyl-d-glucosamine-specific lectin and anti-trefoil factor 2 antibodies, indicating their differentiation into glandular mucus-secreting cells. Conclusions: Microfibrous PCL scaffolds supported growth and differentiation of mGS cells into mucus-secreting cells. These data will help lay groundwork for future experiments to explore use of gastric stem cells and PCL scaffolds in stomach tissue engineering.