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

Abstract

To generate various polycaprolactone (PCL) scaffolds and test their suitability for growth and differentiation of immortalized mouse gastric stem (mGS) cells. 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 3days culture, using viability assay and microscopy. Secondly, growth pattern of the cells on microfibrous scaffolds was studied after 3, 6, 9 and 12days culture using DNA PicoGreen assay and scanning electron microscopy. Thirdly, differentiation of the cells grown on microfibrous scaffolds for 3 and 9days was analysed using lectin/immunohistochemistry. The mGS cells grew preferentially on microfibrous scaffolds. From 3 to 6days, 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 9days 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. 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.